EP3204509B1 - Bioluminescent succinate detection assay - Google Patents

Bioluminescent succinate detection assay Download PDF

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EP3204509B1
EP3204509B1 EP15727175.0A EP15727175A EP3204509B1 EP 3204509 B1 EP3204509 B1 EP 3204509B1 EP 15727175 A EP15727175 A EP 15727175A EP 3204509 B1 EP3204509 B1 EP 3204509B1
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succinate
adp
detection reagent
coa
reaction mixture
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German (de)
English (en)
French (fr)
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EP3204509A1 (en
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Juliano ALVES
Said A. Goueli
Hicham Zegzouti
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Promega Corp
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Promega Corp
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/66—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving luciferase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/25—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving enzymes not classifiable in groups C12Q1/26 - C12Q1/66
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/9015—Ligases (6)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/902—Oxidoreductases (1.)
    • G01N2333/90245—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/91194—Transferases (2.) transferring sulfur containing groups (2.8)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • G01N2333/91205—Phosphotransferases in general
    • G01N2333/91235—Phosphotransferases in general with a phosphate group as acceptor (2.7.4)

Definitions

  • the present invention relates to methods for detecting and quantifying succinate and methods for detecting and quantifying 2-oxoglutarate oxygenases, such as JumonjiC domain-containing histone lysine demethylases and 2-oxoglutarate-dependent dioxygenases.
  • 2-oxoglutarate oxygenases such as JumonjiC domain-containing histone lysine demethylases and 2-oxoglutarate-dependent dioxygenases.
  • PTMs Post translational modifications of proteins are important in determining the epigenetic status of the genome. These modifications include phosphorylation, methylation, acetylation, glycosylation, ubiquitination, nitrosylation, and lipidation and influence many aspects of normal cell biology and pathogenesis. More specifically, histone related PTMs are of great importance as covalent modifications of histones have been implicated in transcriptional regulation via chromatin modulation.
  • post translational modifying enzymes include, but are not limited to, Kinases/Phosphatases, Methyltransferases/Demethylases, Acetyltransferases/Histone Deacetylases, Glycosyltransferases/Glucanases and ADP-Ribosyl Transferases.
  • PTM enzymes Under normal physiological conditions, the regulation of PTM enzymes is tightly regulated. However, under pathological conditions, the activity of these enzymes can be dysregulated, and the disruption of the intracellular networks governed by these enzymes leads to many diseases including cancer and inflammation. Consequently, PTM enzymes have become an important target for drug discovery creating a need for technological development to monitor their activities.
  • These assays can be applied not only to High-Throughput Screening (HTS) to search for drug candidates against these diseases, but also to understand the role of these post-translational modifications in regulating cellular processes.
  • HTS High-Throughput Screening
  • JMJC demethylases JumonjiC domain-containing histone lysine demethylases
  • JMJC demethylases play a role in determining the epigenetic status of the genome by counteracting the activities of histone lysine methyltransferases. These enzymes act as erasers by catalyzing the removal of methyl marks from specific lysine sites in histones, leading to either transcriptional repression or activation of target genes.
  • JMJC demethylases are widely studied and, because of their implication in cancer, they have become validated drug targets. Thus, assays that monitor JMJC demethylase activities are desirable as basic research tools to elucidate their mode of regulation as well as to facilitate the identification of selective and potent inhibitors for drug discovery.
  • JMJC demethylase assays use antibodies, enzyme-coupled assays, HPLC-based assays, or mass spectrometry to detect the substrates that have been demethylated by JMJC demethylases. These assays are not easily configured for rapid demethylase activity detection because they rely on the use of colorimetric, radioactive or fluorimetric non-homogenous antibody-based assays. Colorimetric assays are usually not desirable for drug discovery applications since they are prone to compound interference, low sensitivity, and higher rate of false hits. Radioactivity and mass spectrometry-based methods require sample processing. The use of radiolabeled materials requires waste management or cumbersome regulatory procedures. Because of the importance of these classes of enzymes, there is a need in developing enzymatic assays to monitor their activities, their regulation or identify novel inhibitors.
  • Luo et al. (2006) discloses an assay for Fe(II)/2-oxoglutarate-dependent dioxygenases that uses succinyl-coenzyme A synthetase, pyruvate kinase, and lactate dehydrogenase to couple the formation of the product succinate to the conversion of reduced nicotinamide adenine dinucleotide (NADH) to nicotinamide adenine dinucleotide (NAD).
  • NADH nicotinamide adenine dinucleotide
  • NAD nicotinamide adenine dinucleotide
  • the product succinate is converted to succinyl-CoA, which is subsequently converted to pyruvate and ATP by pyruvate kinase, which further react in the presence of lactate dehydrogenase resulting in the conversion of NADH to NAD.
  • This conversion of NADH to NAD is measured in order to measure succinate, and subsequently the activity of the enzyme.
  • the present invention is directed to a method for luminescent detection or determination of succinate in a sample.
  • the method comprises: (a) contacting the sample with a first detection reagent to form a first reaction mixture; (b) contacting the first reaction mixture with a second detection reagent to form a second reaction mixture; and (c) detecting luminescence in the second reaction mixture, thereby detecting or determining the presence or amount of succinate in the sample; wherein if succinate is present in the sample, the first detection reagent and the second detection reagent converts the succinate to ATP.
  • Contacting the first reaction mixture with a second detection reagent may generate ATP.
  • the bioluminescent enzyme may be a luciferase.
  • the luciferase may be a recombinant luciferase. In some examples the luciferase may be a thermostable and/or a chemostable luciferase. In some examples the luciferase may be a thermostable firefly luciferase.
  • the present invention is directed to a method for detecting or determining the presence or amount of succinate in a sample.
  • the method comprises: (a) contacting the sample with a first detection reagent to form a first reaction mixture; (b) contacting the first reaction mixture with a second detection reagent to form a second reaction mixture; and (c) detecting luminescence in the second reaction mixture, thereby detecting or determining the presence or amount of succinate in the sample; wherein: (i) the first detection reagent comprises 3-oxoacid CoA-transferase (SCOT), inorganic phosphate, and acetoacetyl-CoA and the second detection reagent comprises succinyl-CoA ligase (SCS), ADP, a bioluminescent enzyme, and a luciferin substrate; (ii) the first detection reagent comprises GDP forming succinyl-CoA ligase (SCS-GDP), GTP, and coenzyme A and the second detection reagent
  • the first detection reagent may comprise 3-oxoacid CoA-transferase (SCOT), inorganic phosphate, and acetoacetyl-CoA and the second detection reagent may comprise succinyl-CoA ligase (SCS), ADP, a thermostable firefly luciferase, and D-luciferin.
  • the first detection reagent may comprise GDP forming succinyl-CoA ligase (SCS-GDP), GTP, and coenzyme A and the second detection reagent may comprise guanylate kinase (GMPK), ADP, a thermostable firefly luciferase, and D-luciferin.
  • the first detection reagent may comprise ADP forming succinyl-CoA ligase (SCS-ADP), ATP, and coenzyme A and the second detection reagent may comprise an ATP depletion reagent and subsequently an ADP to ATP conversion/detection reagent, wherein the first reaction mixture is contacted sequentially with the ATP depletion reagent and the ADP to ATP conversion/detection reagent, and wherein the ATP depletion reagent comprises adenylate cyclase and pyrophosphatase and the ADP to ATP conversion/detection reagent comprises pyruvate kinase, phosphoenolpyruvate, a thermostable firefly luciferase, and D-luciferin.
  • SCS-ADP succinyl-CoA ligase
  • ATP succinyl-CoA ligase
  • coenzyme A coenzyme A
  • the succinate forming enzyme may be a 2-oxoglutarate oxygenase.
  • the bioluminescent enzyme may be a luciferase.
  • the luciferase may be a recombinant luciferase.
  • the luciferase may be a thermostable and/or a chemostable luciferase.
  • the luciferase may be a thermostable firefly luciferase.
  • the present invention is directed to a method for detecting or determining the presence or amount of a succinate forming enzyme in a sample or the activity of a succinate forming enzyme.
  • the method comprises: (a) contacting the sample with a peptide, protein, or non-protein substrate, 2-oxoglutarate, Fe (II), and ascorbate to form a succinate reaction mixture, wherein succinate is formed if the sample comprises a succinate forming enzyme; (b) contacting the succinate reaction mixture with a first detection reagent to form a first reaction mixture, wherein the succinate formed in step (a) is converted to succinyl-CoA; (c) contacting the first reaction mixture with a second detection reagent to form a second reaction mixture; and (d) detecting luminescence in the second reaction mixture, thereby detecting or determining the presence or amount of succinate forming enzyme or succinate forming enzyme activity in the sample, wherein: (i) the first detection reagent comprises 3-oxoacid CoA
  • the succinate forming enzyme may be a 2-oxoglutarate oxygenase.
  • the 2-oxoglutarate oxygenase may be a Fe (II) dependent lysine demethylase or a 2-oxoglutarate-dependent dioxygenase.
  • the Fe (II) dependent lysine demethylases may be a JumonjiC domain-containing histone lysine (JMJC) demethylase.
  • JMJC demethylase may be a member of the KDM3, KDM4, KDM5, or KDM6 family of histone demethylases.
  • the JMJC demethylase may be JMJD1A, JMJD1B, JMJD1C, JMJD2A, JMJD2B, JMJD2C, JMJD2D, JMJD3, JMJD4, JMJD5, or JMJD6.
  • the JMJC demethylase may be JMJD2A or JMJD2C.
  • the sample may be contacted with a peptide, protein, or non-protein substrate.
  • the peptide or protein substrate may be a histone peptide substrate.
  • the histone peptide substrate may be histone H3 Me3K9, histone H3 Me2K9, histone H3 Me1K9, histone H3 Me3K4, histone H3 Me2K4, histone H3 Me3K27, histone H3 Me2K27, histone H3 Me3K36, or histone H3 Me2K36.
  • the bioluminescent enzyme may be a luciferase.
  • the luciferase may be a recombinant luciferase.
  • the luciferase may be a thermostable and/or a chemostable luciferase.
  • the luciferase may be a thermostable firefly luciferase.
  • the present invention is directed to a method for determining whether a compound modulates 2-oxoglutarate oxygenase activity in a sample.
  • the method comprises: (a) contacting the sample with the compound to form a test sample, wherein the sample comprises a 2-oxoglutarate oxygenase; (b) contacting the test sample with a peptide, protein, or non-protein substrate, 2-oxoglutarate, Fe (II), and ascorbate to form a succinate reaction mixture; (c) contacting the succinate reaction mixture with 3-oxoacid CoA-transferase (SCOT), inorganic phosphate, and acetoacetyl-CoA to form a first reaction mixture, whereby the succinate formed in step (a) is converted to succinyl-CoA; (d) contacting the first reaction mixture with an ATP detection reagent to form a second reaction mixture, wherein the ATP detection reagent comprises succinyl-CoA ligase (SCS),
  • the 2-oxoglutarate oxygenase may be a Fe (II) dependent lysine demethylase or a 2-oxoglutarate-dependent dioxygenase.
  • the Fe (II) dependent lysine demethylases may be a JumonjiC domain-containing histone lysine (JMJC) demethylase.
  • JMJC demethylase may be a member of the KDM3, KDM4, KDM5, or KDM6 family of histone demethylases.
  • the JMJC demethylase may be JMJD1A, JMJD1B, JMJD1C, JMJD2A, JMJD2B, JMJD2C, JMJD2D, JMJD3, JMJD4, JMJD5, or JMJD6.
  • the JMJC demethylase may be JMJD2A or JMJD2C.
  • the sample may be contacted with a peptide, protein, or non-protein substrate.
  • the peptide or protein substrate may be a histone peptide substrate.
  • the histone peptide substrate may be histone H3 Me3K9, histone H3 Me2K9, histone H3 Me1K9, histone H3 Me3K4, histone H3 Me2K4, histone H3 Me3K27, histone H3 Me2K27, histone H3 Me3K36, or histone H3 Me2K36.
  • the bioluminescent enzyme may be a luciferase.
  • the luciferase may be a recombinant luciferase.
  • the luciferase may be a thermostable and/or a chemostable luciferase.
  • the luciferase may be a thermostable firefly luciferase.
  • the method comprises: (a) contacting the sample with the compound to form a test sample, wherein the sample comprises a 2-oxoglutarate oxygenase; (b) contacting the test sample with a peptide, protein, or non-protein substrate, 2-oxoglutarate, Fe (II), and ascorbate to form a succinate reaction mixture; (c) contacting the succinate reaction mixture with GDP forming succinyl-CoA ligase (SCS-GDP), GTP, and coenzyme A; to form a first reaction mixture, whereby the succinate formed in step (a) is converted to succinyl-CoA and GDP; (d) contacting the first reaction mixture with an ATP detection reagent to form a second reaction mixture, wherein the ATP detection reagent comprises guanylate kinase (GMPK), ADP, a bioluminescent
  • the 2-oxoglutarate oxygenase may be a Fe (II) dependent lysine demethylase or a 2-oxoglutarate-dependent dioxygenase.
  • the Fe (II) dependent lysine demethylases may be a JumonjiC domain-containing histone lysine (JMJC) demethylase.
  • JMJC demethylase may be a member of the KDM3, KDM4, KDM5, or KDM6 family of histone demethylases.
  • the JMJC demethylase may be JMJD1A, JMJD1B, JMJD1C, JMJD2A, JMJD2B, JMJD2C, JMJD2D, JMJD3, JMJD4, JMJD5, or JMJD6.
  • the JMJC demethylase may be JMJD2A or JMJD2C.
  • the sample may be contacted with a peptide, protein, or non-protein substrate.
  • the peptide or protein substrate may be a histone peptide substrate.
  • the histone peptide substrate may be histone H3 Me3K9, histone H3 Me2K9, histone H3 Me1K9, histone H3 Me3K4, histone H3 Me2K4, histone H3 Me3K27, histone H3 Me2K27, histone H3 Me3K36, or histone H3 Me2K36.
  • the bioluminescent enzyme may be a luciferase.
  • the luciferase may be a recombinant luciferase.
  • the luciferase may be a thermostable and/or a chemostable luciferase.
  • the luciferase may be a thermostable firefly luciferase.
  • the present invention is directed to a method for determining whether a compound modulates 2-oxoglutarate oxygenase activity in a sample.
  • the method comprises: (a) contacting the sample with the compound to form a test sample, wherein the sample comprises a 2-oxoglutarate oxygenase; (b) contacting the test sample with a peptide, protein, or non-protein substrate, 2-oxoglutarate, Fe (II), and ascorbate to form a succinate reaction mixture; (c) contacting the succinate reaction mixture with ADP forming succinyl-CoA ligase (SCS-ADP), ATP, and coenzyme A to form a first reaction mixture, whereby the succinate formed in step (a) is converted to succinyl-CoA and ADP; (d) contacting the first reaction mixture with an ATP depletion reagent to form a second reaction mixture, wherein the ATP depletion reagent comprises adenylate cyclase and pyr
  • the 2-oxoglutarate oxygenase may be a Fe (II) dependent lysine demethylase or a 2-oxoglutarate-dependent dioxygenase.
  • the Fe (II) dependent lysine demethylases may be a JumonjiC domain-containing histone lysine (JMJC) demethylase.
  • JMJC demethylase may be a member of the KDM3, KDM4, KDM5, or KDM6 family of histone demethylases.
  • the JMJC demethylase may be JMJD1A, JMJD1B, JMJD1C, JMJD2A, JMJD2B, JMJD2C, JMJD2D, JMJD3, JMJD4, JMJD5, or JMJD6.
  • the JMJC demethylase may be JMJD2A or JMJD2C.
  • the sample may be contacted with a peptide, protein, or non-protein substrate.
  • the peptide or protein substrate may be a histone peptide substrate.
  • the histone peptide substrate may be histone H3 Me3K9, histone H3 Me2K9, histone H3 Me1K9, histone H3 Me3K4, histone H3 Me2K4, histone H3 Me3K27, histone H3 Me2K27, histone H3 Me3K36, or histone H3 Me2K36.
  • the bioluminescent enzyme may be a luciferase.
  • the luciferase may be a recombinant luciferase.
  • the luciferase may be a thermostable and/or a chemostable luciferase.
  • the luciferase may be a thermostable firefly luciferase.
  • the present invention is directed to a kit for detecting succinate in a sample.
  • the kit comprises: (i) 3-oxoacid CoA-transfer (SCOT); (ii) inorganic phosphate; (iii) acetoacetyl-CoA; (iv) succinyl-CoA ligase (SCS); (v) ADP; (vi) a bioluminescent enzyme; and (vii) a luciferin substrate, wherein (i)-(vii) are in one or more containers.
  • SCS succinyl-CoA ligase
  • the kit may comprise a first detection reagent comprising: (i) 3-oxoacid CoA-transfer (SCOT); (ii) inorganic phosphate; and (iii) acetoacetyl-CoA; and a second detection reagent comprising: (iv) succinyl-CoA ligase (SCS); (v) ADP; (vi) a bioluminescent enzyme; and (vii) a luciferin substrate.
  • the bioluminescent enzyme may be a thermostable firefly luciferase and the luciferin substrate may be D-luciferin.
  • the kit may further comprise instructions for using the kit to detect or determine the presence or amount of succinate in the sample.
  • the bioluminescent enzyme may be a luciferase.
  • the luciferase may be a recombinant luciferase.
  • the luciferase may be a thermostable and/or a chemostable luciferase.
  • the luciferase may be a thermostable firefly luciferase.
  • the present invention is directed to a kit for detecting succinate in a sample.
  • the kit comprises: (i) GDP forming succinyl-CoA ligase (SCS-GDP); (ii) GTP; (iii) coenzyme A; (iv) guanylate kinase (GMPK); (v) ADP; (vi) a bioluminescent enzyme; and (vii) a luciferin substrate, wherein (i)-(vii) are in one or more containers.
  • the kit may comprise a first detection reagent comprising: (i) GDP forming succinyl-CoA ligase (SCS-GDP); (ii) GTP; and (iii) coenzyme A; and a second detection reagent comprising: (iv) guanylate kinase (GMPK); (v) ADP; (vi) a bioluminescent enzyme; and (vii) a luciferin substrate.
  • the kit may further comprise instructions for using the kit to detect or determine the presence or amount of succinate in the sample.
  • the bioluminescent enzyme may be a luciferase.
  • the luciferase may be a recombinant luciferase.
  • the luciferase may be a thermostable and/or a chemostable luciferase.
  • the luciferase may be a thermostable firefly luciferase.
  • the present invention is directed to a kit for detecting succinate in a sample.
  • the kit comprises: (i) ADP forming succinyl-CoA ligase (SCS-ADP); (ii) ATP; (iii) coenzyme A; (iv) adenylate cyclase; (v) pyrophosphatase; (vi) pyruvate kinase; (vii) phosphoenolpyruvate; (viii) a bioluminescent enzyme; and (ix) a luciferin substrate, wherein (i)-(ix) are in one or more containers.
  • SCS-ADP succinyl-CoA ligase
  • the first detection reagent may comprise: (i) ADP forming succinyl-CoA ligase (SCS-ADP); (ii) ATP; and (iii) coenzyme A; and the second detection reagent may comprise an ATP depletion reagent and an ADP to ATP conversion/detection reagent, wherein the ATP depletion reagent may comprise: (iv) adenylate cyclase; and (v) pyrophosphatase; and the ADP to ATP conversion/detection reagent may comprise: (vi) pyruvate kinase; (vii) phosphoenolpyruvate; (viii) a bioluminescent enzyme; and (ix) a luciferin substrate.
  • SCS-ADP succinyl-CoA ligase
  • ATP depletion reagent may comprise: (iv) adenylate cyclase; and (v) pyrophosphata
  • the kit may further comprise instructions for using the kit to detect or determine the presence or amount of succinate in the sample.
  • the bioluminescent enzyme may be a luciferase.
  • the luciferase may be a recombinant luciferase.
  • the luciferase may be a thermostable and/or a chemostable luciferase.
  • the luciferase may be a thermostable firefly luciferase.
  • the present invention is directed to a kit for detecting or determining 2-oxoglutarate oxygenase activity in a sample.
  • the kit comprises: (i) a peptide, protein, or non-protein substrate; (ii) 2-oxoglutarate; (iii) Fe (II); (iv) ascorbate; (v) 3-oxoacid CoA-transfer (SCOT); (vi) inorganic phosphate; (vii) acetoacetyl-CoA; (viii) succinyl-CoA ligase (SCS); (ix) ADP; (x) a bioluminescent enzyme; and (xi) a luciferin substrate, wherein (i)-(xi) are in one or more containers.
  • the kit may comprise a first detection reagent comprising: (v) 3-oxoacid CoA-transfer (SCOT); (vi) inorganic phosphate; and (vii) acetoacetyl-CoA; and a second detection reagent comprising: (viii) succinyl-CoA ligase (SCS); (ix) ADP; (x) a bioluminescent enzyme; and (xi) a luciferin substrate. Also disclosed herein, the kit may further comprise instructions for using the kit to detect or determine 2-oxoglutarate oxygenase activity in a sample.
  • a first detection reagent comprising: (v) 3-oxoacid CoA-transfer (SCOT); (vi) inorganic phosphate; and (vii) acetoacetyl-CoA
  • a second detection reagent comprising: (viii) succinyl-CoA ligase (SCS); (ix) ADP; (x) a bioluminescent enzyme
  • the 2-oxoglutarate oxygenase may be a Fe (II) dependent lysine demethylase or a 2-oxoglutarate-dependent dioxygenase.
  • the Fe (II) dependent lysine demethylases may be a JumonjiC domain-containing histone lysine (JMJC) demethylase.
  • JMJC demethylase may be a member of the KDM3, KDM4, KDM5, or KDM6 family of histone demethylases.
  • the JMJC demethylase may be JMJD1A, JMJD1B, JMJD1C, JMJD2A, JMJD2B, JMJD2C, JMJD2D, JMJD3, JMJD4, JMJD5, or JMJD6.
  • the JMJC demethylase may be JMJD2A or JMJD2C.
  • the kit may comprise a peptide, protein, or non-protein substrate.
  • the peptide or protein substrate may be a histone peptide substrate.
  • the histone peptide substrate may be histone H3 Me3K9, histone H3 Me2K9, histone H3 Me1K9, histone H3 Me3K4, histone H3 Me2K4, histone H3 Me3K27, histone H3 Me2K27, histone H3 Me3K36, or histone H3 Me2K36.
  • the bioluminescent enzyme may be a luciferase.
  • the luciferase may be a recombinant luciferase.
  • the luciferase may be a thermostable and/or a chemostable luciferase.
  • the luciferase may be a thermostable firefly luciferase.
  • the present invention is directed to a kit for detecting or determining 2-oxoglutarate oxygenase activity in a sample.
  • the kit comprises: (i) a peptide, protein, or non-protein substrate; (ii) 2-oxoglutarate; (iii) Fe (II); (iv) ascorbate; (v) GDP forming succinyl-CoA ligase (SCS-GDP); (vi) GTP; (vii) coenzyme A; (viii) guanylate kinase (GMPK); (ix) ADP; (x) a bioluminescent enzyme; and (xi) a luciferin substrate, wherein (i)-(xi) are in one or more containers.
  • the kit may comprise a first detection reagent comprising: (v) GDP forming succinyl-CoA ligase (SCS-GDP); (vi) GTP; and (vii) coenzyme A; and a second detection reagent comprising: (viii) guanylate kinase (GMPK); (ix) ADP; (x) a bioluminescent enzyme; and (xi) a luciferin substrate. Also disclosed herein, the kit may further comprise instructions for using the kit to detect or determine 2-oxoglutarate oxygenase activity in a sample.
  • SCS-GDP succinyl-CoA ligase
  • GTP GTP
  • coenzyme A coenzyme A
  • a second detection reagent comprising: (viii) guanylate kinase (GMPK); (ix) ADP; (x) a bioluminescent enzyme; and (xi) a luciferin substrate.
  • the kit may further comprise instructions for using the
  • the 2-oxoglutarate oxygenase may be a Fe (II) dependent lysine demethylase or a 2-oxoglutarate-dependent dioxygenase.
  • the Fe (II) dependent lysine demethylases may be a JumonjiC domain-containing histone lysine (JMJC) demethylase.
  • JMJC demethylase may be a member of the KDM3, KDM4, KDM5, or KDM6 family of histone demethylases.
  • the JMJC demethylase may be JMJD1A, JMJD1B, JMJD1C, JMJD2A, JMJD2B, JMJD2C, JMJD2D, JMJD3, JMJD4, JMJD5, or JMJD6.
  • the JMJC demethylase may be JMJD2A or JMJD2C.
  • the kit may comprise a peptide, protein, or non-protein substrate.
  • the peptide or protein substrate may be a histone peptide substrate.
  • the histone peptide substrate may be histone H3 Me3K9, histone H3 Me2K9, histone H3 Me1K9, histone H3 Me3K4, histone H3 Me2K4, histone H3 Me3K27, histone H3 Me2K27, histone H3 Me3K36, or histone H3 Me2K36.
  • the bioluminescent enzyme may be a luciferase.
  • the luciferase may be a recombinant luciferase.
  • the luciferase may be a thermostable and/or a chemostable luciferase.
  • the luciferase may be a thermostable firefly luciferase.
  • the present invention is directed to a kit for detecting or determining 2-oxoglutarate oxygenase activity in a sample.
  • the kit comprises: (i) a peptide, protein, or non-protein substrate; (ii) 2-oxoglutarate; (iii) Fe (II); (iv) ascorbate; (v) ADP forming succinyl-CoA ligase (SCS-ADP); (vi) ATP; (vii) coenzyme A; (viii) adenylate cyclase; (ix) pyrophosphatase; (x) pyruvate kinase; (xi) phosphoenolpyruvate; (xii) a bioluminescent enzyme; and (xiii) a luciferin substrate, wherein (i)-(xiii) are in one or more containers.
  • the kit may comprise a first detection reagent comprising: (v) ADP forming succinyl-CoA ligase (SCS-ADP); (vi) ATP; and (vii) coenzyme A; and a second detection reagent comprising an ATP depletion reagent and an ADP to ATP conversion/detection reagent, wherein the ATP depletion reagent may comprise: (viii) adenylate cyclase; and (ix) pyrophosphatase; and the ADP to ATP conversion/detection reagent may comprise: (x) pyruvate kinase; (xi) phosphoenolpyruvate; (xii) a bioluminescent enzyme; and (xiii) a luciferin substrate.
  • a first detection reagent comprising: (v) ADP forming succinyl-CoA ligase (SCS-ADP); (vi) ATP; and (vii)
  • the kit may further comprise instructions for using the kit to detect or determine 2-oxoglutarate oxygenase activity in a sample.
  • the 2-oxoglutarate oxygenase may be a Fe (II) dependent lysine demethylase or a 2-oxoglutarate-dependent dioxygenase.
  • the Fe (II) dependent lysine demethylases may be a JumonjiC domain-containing histone lysine (JMJC) demethylase.
  • JMJC demethylase may be a member of the KDM3, KDM4, KDM5, or KDM6 family of histone demethylases.
  • the JMJC demethylase may be JMJD1A, JMJD1B, JMJD1C, JMJD2A, JMJD2B, JMJD2C, JMJD2D, JMJD3, JMJD4, JMJD5, or JMJD6.
  • the JMJC demethylase may be JMJD2A or JMJD2C.
  • the kit may comprise a peptide, protein, or non-protein substrate.
  • the peptide or protein substrate may be a histone peptide substrate.
  • the histone peptide substrate may be histone H3 Me3K9, histone H3 Me2K9, histone H3 Me1K9, histone H3 Me3K4, histone H3 Me2K4, histone H3 Me3K27, histone H3 Me2K27, histone H3 Me3K36, or histone H3 Me2K36.
  • the bioluminescent enzyme may be a luciferase.
  • the luciferase may be a recombinant luciferase.
  • the luciferase may be a thermostable and/or a chemostable luciferase.
  • the luciferase may be a thermostable firefly luciferase.
  • the present invention is directed to a method for luminescent detection or determination of succinate in a sample, the method comprising: (a) contacting the sample with a first detection reagent to form a first reaction mixture; (b) contacting the first reaction mixture with a second detection reagent to form a second reaction mixture; and (c) detecting luminescence in the second reaction mixture, thereby detecting or determining the presence or amount of succinate in the sample; wherein if succinate is present in the sample, the first detection reagent and the second detection reagent converts the succinate to ATP.
  • the present invention is directed to a method for detecting or determining the presence or amount of succinate in a sample, the method comprising: (a) contacting the sample with a first detection reagent to form a first reaction mixture; (b) contacting the first reaction mixture with a second detection reagent to form a second reaction mixture; and (c) detecting luminescence in the second reaction mixture, thereby detecting or determining the presence or amount of succinate in the sample; wherein: (i) the first detection reagent comprises 3-oxoacid CoA-transferase (SCOT), inorganic phosphate, and acetoacetyl-CoA and the second detection reagent comprises succinyl-CoA ligase (SCS), ADP, a bioluminescent enzyme, and a luciferin substrate; (ii) the first detection reagent comprises 3-oxoacid CoA-transferase (SCOT), inorganic phosphate, acetoacetyl-CoA, succin
  • the present invention is directed to a method for detecting or determining the presence or amount of a succinate forming enzyme in a sample or the activity of a succinate forming enzyme, the method comprising: (a) contacting the sample with a peptide, protein, or non-protein substrate, 2-oxoglutarate, Fe(II), and ascorbate to form a succinate reaction mixture, wherein succinate is formed if the sample comprises a succinate forming enzyme; (b) contacting the succinate reaction mixture with a first detection reagent to form a first reaction mixture, wherein the succinate formed in step (a) is converted to succinyl-CoA; (c) contacting the first reaction mixture with a second detection reagent to form a second reaction mixture; and (d) detecting luminescence in the second reaction mixture, thereby detecting or determining the presence or amount of succinate forming enzyme or succinate forming enzyme activity in the sample, wherein: (i) the first detection reagent comprises 3-oxoacid CoA-
  • the present invention is directed to a method for determining whether a compound modulates 2-oxoglutarate oxygenase activity in a sample, the method comprising: (a) contacting the sample with the compound to form a test sample, wherein the sample comprises a 2-oxoglutarate oxygenase; (b) contacting the test sample with a peptide, protein, or non-protein substrate, 2-oxoglutarate, Fe(II), and ascorbate to form a succinate reaction mixture; (c) contacting the succinate reaction mixture with: (i) 3-oxoacid CoA-transferase (SCOT), inorganic phosphate, and acetoacetyl-CoA to form a first reaction mixture, whereby the succinate formed in step (a) is converted to succinyl-CoA; or (ii) 3-oxoacid CoA-transferase (SCOT), inorganic phosphate, acetoacetyl-CoA, succinyl-Co
  • the present invention is directed to a method for determining whether a compound modulates 2-oxoglutarate oxygenase activity in a sample, the method comprising: (a) contacting the sample with the compound to form a test sample, wherein the sample comprises a 2-oxoglutarate oxygenase; (b) contacting the test sample with a peptide, protein, or non-protein substrate, 2-oxoglutarate, Fe(II), and ascorbate to form a succinate reaction mixture; (c) contacting the succinate reaction mixture with GDP forming succinyl-CoA ligase (SCS-GDP), GTP, and coenzyme A; to form a first reaction mixture, whereby the succinate formed in step (a) is converted to succinyl-CoA and GDP; (d) contacting the first reaction mixture with an ATP detection reagent to form a second reaction mixture, wherein the ATP detection reagent comprises guanylate kinase (GMPK), ADP, a bio
  • the present invention is directed to a method for determining whether a compound modulates 2-oxoglutarate oxygenase activity in a sample, the method comprising: (a) contacting the sample with the compound to form a test sample, wherein the sample comprises a 2-oxoglutarate oxygenase; (b) contacting the test sample with a peptide, protein, or non-protein substrate, 2-oxoglutarate, Fe(II), and ascorbate to form a succinate reaction mixture; (c) contacting the succinate reaction mixture with ADP forming succinyl-CoA ligase (SCS-ADP), ATP, and coenzyme A to form a first reaction mixture, whereby the succinate formed in step (a) is converted to succinyl-CoA and ADP; (d) contacting the first reaction mixture with an ATP depletion reagent to form a second reaction mixture, wherein the ATP depletion reagent comprises adenylate cyclase and pyro
  • the present invention is directed to a kit for detecting succinate in a sample, the kit comprising: (i) 3-oxoacid CoA-transferase (SCOT); (ii) inorganic phosphate; (iii) acetoacetyl-CoA; (iv) succinyl-CoA ligase (SCS); (v) ADP; (vi) a bioluminescent enzyme; and (vii) a luciferin substrate, wherein (i)-(vii) are in one or more containers.
  • SCS succinyl-CoA ligase
  • the present invention is directed to a kit for detecting succinate in a sample, the kit comprising: (i) GDP forming succinyl-CoA ligase (SCS-GDP); (ii) GTP; (iii) coenzyme A; (iv) guanylate kinase (GMPK); (v) ADP; (vi) a bioluminescent enzyme; and (vii) a luciferin substrate, wherein (i)-(vii) are in one or more containers.
  • SCS-GDP succinyl-CoA ligase
  • GTP GTP
  • coenzyme A coenzyme A
  • GMPK guanylate kinase
  • ADP a bioluminescent enzyme
  • a bioluminescent enzyme a bioluminescent enzyme
  • luciferin substrate wherein (i)-(vii) are in one or more containers.
  • the present invention is directed to a kit for detecting succinate in a sample, the kit comprising: (i) ADP forming succinyl-CoA ligase (SCS-ADP); (ii) ATP; (iii) coenzyme A; (iv) adenylate cyclase; (v) pyrophosphatase; (vi) pyruvate kinase; (vii) phosphoenolpyruvate; (viii) a bioluminescent enzyme; and (ix) a luciferin substrate, wherein (i)-(ix) are in one or more containers.
  • SCS-ADP succinyl-CoA ligase
  • the present invention is directed to a kit for detecting or determining 2-oxoglutarate oxygenase activity in a sample, the kit comprising: (i) a peptide, protein, or non-protein substrate; (ii) 2-oxoglutarate; (iii) Fe(II); (iv) ascorbate; (v) 3-oxoacid CoA-transferase (SCOT); (vi) inorganic phosphate; (vii) acetoacetyl-CoA; (viii) succinyl-CoA ligase (SCS); (ix) ADP; (x) a bioluminescent enzyme; and (xi) a luciferin substrate, wherein (i)-(xi) are in one or more containers.
  • a kit for detecting or determining 2-oxoglutarate oxygenase activity in a sample comprising: (i) a peptide, protein, or non-protein substrate; (ii) 2-oxoglutarate
  • the present invention is directed to a kit for detecting or determining 2-oxoglutarate oxygenase activity in a sample, the kit comprising: (i) a peptide, protein, or non-protein substrate; (ii) 2-oxoglutarate; (iii) Fe(II); (iv) ascorbate; (v) GDP forming succinyl-CoA ligase (SCS-GDP); (vi) GTP; (vii) coenzyme A; (viii) guanylate kinase (GMPK); (ix) ADP; (x) a bioluminescent enzyme; and (xi) a luciferin substrate, wherein (i)-(xi) are in one or more containers.
  • the present invention is directed to a kit for detecting or determining 2-oxoglutarate oxygenase activity in a sample, the kit comprising: (i) a peptide, protein, or non-protein substrate; (ii) 2-oxoglutarate; (iii) Fe(II); (iv) ascorbate; (v) ADP forming succinyl-CoA ligase (SCS-ADP); (vi) ATP; (vii) coenzyme A; (viii) adenylate cyclase; (ix) pyrophosphatase; (x) pyruvate kinase; (xi) phosphoenolpyruvate; (xii) a bioluminescent enzyme; and (xiii) a luciferin substrate, wherein (i)-(xiii) are in one or more containers.
  • Also disclosed herein is a method for detecting or determining the presence or amount of succinate in a sample, the method comprising: (a) contacting the sample with a first detection reagent to form a first reaction mixture; (b) contacting the first reaction mixture with a second detection reagent to form a second reaction mixture; and (c) detecting luminescence in the second reaction mixture, thereby detecting or determining the presence or amount of succinate in the sample; wherein the first detection reagent comprises ADP forming succinyl-CoA ligase (SCS-ADP), ATP, and coenzyme A, and the second detection reagent comprises an ATP depletion reagent and subsequently an ADP-to-ATP conversion/detection reagent, wherein the first reaction mixture is contacted sequentially with the ATP depletion reagent and the ADP-to-ATP conversion/detection reagent; and a bioluminescent enzyme, and a luciferin substrate.
  • SCS-ADP
  • the present invention is directed to a method for determining whether a compound modulates 2-oxoglutarate oxygenase activity in a sample, the method comprising: (a) contacting the sample with the compound to form a test sample, wherein the sample comprises a 2-oxoglutarate oxygenase; (b) contacting the test sample with a peptide, protein, or non-protein substrate, 2-oxoglutarate, Fe(II), and ascorbate to form a succinate reaction mixture; (c) contacting the succinate reaction mixture with ADP forming succinyl-CoA ligase (SCS-ADP), ATP, and coenzyme A to form a first reaction mixture, whereby the succinate formed in step (a) is converted to succinyl-CoA and ADP; (d) contacting the first reaction mixture with an ATP depletion reagent to form a second reaction mixture; (e) contacting the second reaction mixture with an ADP-to-ATP conversion/detection reagent
  • kits for detecting succinate in a sample comprising: (i) ADP forming succinyl-CoA ligase (SCS-ADP); (ii) ATP; (iii) coenzyme A; (iv) an ATP depletion reagent; (v) an ADP-to- ATP conversion/detection reagent; (vi) a bioluminescent enzyme; and (vii) a luciferin substrate, wherein (i)-(vii) are in one or more containers.
  • SCS-ADP succinyl-CoA ligase
  • kits for detecting or determining 2-oxoglutarate oxygenase activity in a sample comprising: (i) a peptide, protein, or non-protein substrate; (ii) 2-oxoglutarate; (iii) Fe(II); (iv) ascorbate; (v) ADP forming succinyl-CoA ligase (SCS-ADP); (vi) ATP; (vii) coenzyme A; (viii) an ATP depletion reagent; (ix) an ATP-to-ATP conversion/detection reagent; (x) a bioluminescent enzyme; and (xi) a luciferin substrate, wherein (i)-(xi) are in one or more containers.
  • a versatile bioluminescent and homogeneous succinate detection assay which may be adapted to measure 2-oxoglutarate oxygenase activity, such as JMJC demethylase activity.
  • the bioluminescent succinate detection assay involves 3-oxoacid CoA transferase and/or succinyl-CoA ligase reactions ( Fig. 1 ) to detect succinate in a bioluminescence format (see e.g., Figs. 1 , 8 , 12 , and 16 ).
  • the bioluminescent succinate detection assay detects succinate, it may be used with different substrates in diverse enzyme systems.
  • the bioluminescent succinate detection assay may be used with a succinate formation reaction, such as a demethylation reaction, to detect the presence and/or activity of a succinate forming or generating enzyme, such as 2-oxoglutarate oxygenase (e.g., JMJC demethylase and 2-oxoglutarate-dependent (20G-dependent) dioxygenase).
  • a succinate forming or generating enzyme such as 2-oxoglutarate oxygenase (e.g., JMJC demethylase and 2-oxoglutarate-dependent (20G-dependent) dioxygenase).
  • 2-oxoglutarate oxygenase e.g., JMJC demethylase and 2-oxoglutarate-dependent (20G-dependent) dioxygenase.
  • the assay may also be used for monitoring enzymes involved in DNA demethylation, such as Ten-Eleven Translocation (TET) enzymes.
  • the bioluminescent succinate detection assay has the advantage of detecting 2-oxoglutarate oxygenases and their activities in a two-step assay with short incubation times and a robust and stable signal. This homogeneous assay is fast, sensitive, and simple, and it does not require washing steps.
  • the two-step format assay involves the conversion of the succinate product to ATP, which is measured in a robust luciferase reaction, wherein the light output of the luciferase reaction is proportional to the succinate concentration from low nM to 20 ⁇ M.
  • This highly sensitive assay allows the detection of low activity JMJC demethylases or the use of low amounts of purified JMJC demethylase, 2-oxoglutarate, and/or Fe(II).
  • the assay is highly sensitive and robust, which allows for measuring the activity of a majority of JMJC demethylase subfamilies.
  • the disclosed assays and methods are convenient and may be used with any instrumentation platform without sample processing.
  • the bioluminescent reaction has sufficient sensitivity and steady signal for use in high-throughput screening (HTS).
  • the reagents may be designed with relative ease and may be synthesized readily.
  • the reagents may facilitate measurement of enzymatic activity in many samples in a high throughput format over a long period of time due to the high signal stability generated by a luminogenic reaction, thus eliminating the need for a luminometer with reagent injectors and allowing for batch-mode processing of multiple samples.
  • the present methods may be performed in a single well or in a multi-well plate, making them suitable for use as high throughput screening (HTS) methods for high sample processing or inhibitor screening.
  • HTS high throughput screening
  • the bioluminescent succinate detection assay is a simple-to-use method that allows significant savings of enzyme usage and does not require radioactivity, antibodies, or modified substrates.
  • the bioluminescent succinate detection assay is suitable for studying substrate specificity, evaluating kinetic parameters, and mode of action of JMJC demethylase inhibitors.
  • each intervening number there between with the same degree of precision is explicitly contemplated.
  • the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.
  • 2-oxoglutarate refers to the anion of ⁇ -ketoglutaric acid.
  • 2-oxoglutarate is the keto acid produced by deamination of glutamate, and is an intermediate in the Krebs cycle.
  • 3-oxoacid CoA-transferase and "SCOT” as used interchangeably herein refer to an enzyme that catalyzes the chemical reaction: succinate + acetoacetyl-CoA ⁇ succinyl-CoA + acetoacetate.
  • SCOT is also known as succinyl-CoA:3-oxo-acid CoA-transferase, 3-oxoacid coenzyme A-transferase, 3-ketoacid CoA-transferase, 3-ketoacid coenzyme A transferase, 3-oxoacid CoA dehydrogenase, acetoacetate succinyl-CoA transferase, acetoacetyl coenzyme A-succinic thiophorase, succinyl coenzyme A-acetoacetyl coenzyme A-transferase, and succinyl-CoA transferase.
  • SCOT participates in 3 metabolic pathways: synthesis and degradation of ketone bodies, valine, leucine and isoleucine degradation, and butanoate metabolism.
  • SCOT belongs to the family of CoA-transferases.
  • the term "luminescent” as used herein includes bioluminescence (i.e., light produced by a living organism).
  • bioluminescence i.e., light produced by a living organism.
  • the enzyme involved has evolved in an organism by natural selection for the purpose of generating light, or the enzyme involved is a mutated derivative of such an enzyme, the luminescent reactions are also called “bioluminescent reactions” and the enzyme involved is also called a “bioluminescent enzyme.”
  • bioluminescent enzymes include, without limitation, beetle luciferase, e.g., firefly luciferase, and the like.
  • luciferin substrate refers to a molecule capable of creating light via a chemical or biochemical reaction (e.g., luciferin, luciferin derivative, or a functional analog thereof).
  • the luciferin substrate may be a molecule capable of creating light generated by a luciferase.
  • Suitable luciferin substrates for luciferase enzymes include luciferin, luciferin derivatives, and functional analogs of luciferins.
  • functional analogs of luciferins include modified luciferins including derivatives of these compounds. Exemplary compounds include those disclosed in US Patent Publication No. 2009/0075309 .
  • luciferin derivative refers to a type of luminogenic molecule or compound or luciferin substrate having a substantial structure of D-luciferin and is a luciferase substrate, e.g., aminoluciferin or fluoroluciferin , or luciferase substrates disclosed in U.S. Patent Publication No. 2007/0015790 , Branchini et al. (1989), e.g., naphthyl and quinolyl derivatives, Branchini et al. (2002), and Branchini (2000).
  • Modulate as used herein may mean any altering of activity, such as regulate, down regulate, upregulate, reduce, inhibit, increase, decrease, deactivate, or activate.
  • Series of calibrating compositions refers to a plurality of compositions comprising a known concentration of succinate, wherein each of the compositions differs from the other compositions in the series by the concentration of succinate.
  • Small molecules as used herein may mean a molecule usually less than about 10 kDa molecular weight. Small molecules may be synthetic organic or inorganic compounds, peptides, (poly)nucleotides, (oligo)saccharides and the like. Small molecules specifically include small non-polymeric (i.e., not peptide or polypeptide) organic and inorganic molecules. Many pharmaceutical companies have extensive libraries of such molecules, which may be conveniently screened by using the herein described methods. Small molecules may have molecular weights of less than about 1000 Da, about 750 Da, or about 500 Da.
  • succinyl-CoA synthase succinyl-CoA ligase
  • SCS succinyl-CoA ligase
  • Pi denotes inorganic phosphate
  • NDP denotes nucleoside diphosphate (e.g., GDP or ADP)
  • NTP denotes nucleoside triphosphate (e.g., GTP or ATP).
  • the NDP may also denote CDP, TDP, or UDP.
  • the NTP may also denote CTP, TTP, or UTP.
  • Succinyl-CoA synthase facilitates the coupling of the conversion of succinyl CoA to succinate with the formation of a nucleoside triphosphate molecule, e.g., GTP or ATP, from an inorganic phosphate molecule and a nucleoside diphosphate molecule, e.g., GDP or ADP.
  • a nucleoside triphosphate molecule e.g., GTP or ATP
  • SCS is also known as succinyl-CoA synthetase, succinate-CoA ligase, SUCL, succinic thiokinase, A-STK, G-STK, A-SCS, G-SCS, SCACT, VEG239, VEG63, Vegetative protein 239, Vegetative protein 63, CG11963 and succinate thiokinase.
  • SCS is one of the catalysts involved in the citric acid cycle, a central pathway in cellular metabolism, and it is located within the mitochondrial matrix of a cell.
  • ADP-forming succinyl-CoA synthase refers to a SCS that catalyzes the reaction: ATP + succinate + CoA ⁇ ADP + phosphate + succinyl-CoA.
  • GDP-forming succinyl-CoA synthase refers to a SCS that catalyzes the reaction: GTP + succinate + CoA ⁇ GDP + phosphate + succinyl-CoA.
  • Variant is used herein to describe a peptide or polypeptide that differs in amino acid sequence by the insertion, deletion, or conservative substitution of amino acids, but retain at least one biological activity. Variant is also used herein to describe a protein with an amino acid sequence that is substantially identical to a referenced protein with an amino acid sequence that retains at least one biological activity. A conservative substitution of an amino acid, i.e., replacing an amino acid with a different amino acid of similar properties (e.g., hydrophilicity, degree, and distribution of charged regions) is recognized in the art as typically involving a minor change. "Variant” also can be used to describe a polypeptide or a fragment thereof that has been differentially processed, such as by proteolysis, phosphorylation, or other post-translational modification, yet retains its biological activity.
  • the present disclosure provides a bioluminescent succinate detection assay for detecting succinate in a sample.
  • the succinate detection assay involves the conversion of succinate in the sample to succinyl-CoA using 3-oxoacid CoA-transferase ("SCOT") and/or succinyl-CoA synthase (“SCS”), such as ADP-forming succinyl-CoA synthase (“SCS-ADP”) and GDP-forming succinyl-CoA synthase (“SCS-GDP”).
  • STT 3-oxoacid CoA-transferase
  • SCS succinyl-CoA synthase
  • SCS-ADP ADP-forming succinyl-CoA synthase
  • SCS-GDP succinyl-GDP
  • the enzyme SCOT catalyzes the conversion of succinate and acetoacetyl-CoA into acetoacetate and succinyl-CoA (see Fig. 1 ).
  • the intermediate succinyl-CoA in the presence of ADP and inorganic phosphate, is converted into succinate, CoA, and ATP by the enzyme SCS-ADP in a second reaction (see Fig. 1 ).
  • the ATP that is generated is simultaneously converted into a bioluminescent signal, e.g., bioluminescence using an ATP detection reagent, e.g., a luciferin-luciferase reaction (See Fig. 2 ).
  • the conversion of succinate to succinyl-CoA is performed using a reagent that contains SCOT, acetoacetyl-CoA, potassium phosphate (monobasic/dibasic), and a buffer such as MOPS, Tris or HEPES.
  • the conversion of ADP to ATP is performed separately using a reagent, e.g., an ADP-to-ATP conversion reagent, such as Bioluminescent Succinate Detection Reagent II, which contains SCS (ADP forming), ADP, and magnesium sulfate or other divalent cation (such as magnesium chloride).
  • the ATP may then be measured by the addition of an ATP detection reagent, e.g., a luciferin-luciferase assay reagent, e.g., the Kinase-Glo® assay reagent (Promega Corp.).
  • an ATP detection reagent e.g., a luciferin-luciferase assay reagent, e.g., the Kinase-Glo® assay reagent (Promega Corp.).
  • the amount of luminescence generated is proportional to the amount of succinate present in the sample.
  • the first detection reagent may include SCOT, inorganic phosphate, such as potassium phosphate (monobasic/dibasic), acetoacetyl-CoA (also known as 3-oxoacyl-CoA), and a buffer, such as MOPS, Tris or HEPES.
  • the SCOT enzyme may be a SCOT enzyme from a eukaryote or a prokaryote.
  • the SCOT enzyme may be a SCOT enzyme from Sus scrofa.
  • the SCOT enzyme may be encoded by a 3-oxoacid CoA-transferase gene sequence from a eukaryote or a prokaryote.
  • the SCOT enzyme may be encoded by a 3-oxoacid CoA-transferase gene sequence from Sus scrofa.
  • the first detection reagent may include from about 0.001 ng/ ⁇ L to about 500.00 ng/ ⁇ L, about 0.010 ng/ ⁇ L to about 500.00 ng/ ⁇ L, about 0.10 ng/ ⁇ L to about 500.00 ng/ ⁇ L, about 1.0 ng/ ⁇ L to about 500.00 ng/ ⁇ L, about 10.0 ng/ ⁇ L to about 500.00 ng/ ⁇ L, about 100.00 ng/ ⁇ L to about 500.00 ng/ ⁇ L, about 0.001 ng/ ⁇ L to about 400.00 ng/ ⁇ L, about 0.010 ng/ ⁇ L to about 400.00 ng/ ⁇ L, about 0.10 ng/ ⁇ L to about 400.00 ng/ ⁇ L, about 1.0 ng/ ⁇ L to about 400.00 ng/ ⁇ L, about 10.0 ng/ ⁇ L to about 400.00 ng/ ⁇ L, about 100.00 ng/ ⁇ L to about 400.00 ng/ ⁇ L, about 0.001 ng/ ⁇ L to about 300.00 ng/ ⁇ L, about 0.010 ng
  • the first detection reagent may include from about 50 mM to about 1000 mM, from about 50 mM to about 750 mM, from about 50 mM to about 500 mM, from about 50 mM to about 250 mM, about 100 mM to about 1000 mM, from about 100 mM to about 750 mM, from about 100 mM to about 500 mM, from about 100 mM to about 250 mM, about 250 mM to about 1000 mM, from about 250 mM to about 750 mM, from about 250 mM to about 500 mM, about 500 mM to about 1000 mM, or from about 500 mM to about 750 mM, or at least about 50 mM, at least about 100 mM, at least about 150 mM, at least about 200 mM, at least about 250 mM, at least about 300 mM, at least about 350 mM, at least about 400 mM, at least about 450 mM, at
  • the first detection reagent may include from about 1 ⁇ M to about 1000 ⁇ M, about 1 ⁇ M to about 900 ⁇ M, about 1 ⁇ M to about 800 ⁇ M, about 1 ⁇ M to about 700 ⁇ M, about 1 ⁇ M to about 600 ⁇ M, about 1 ⁇ M to about 500 ⁇ M, about 1 ⁇ M to about 400 ⁇ M, about 1 ⁇ M to about 300 ⁇ M, about 1 ⁇ M to about 200 ⁇ M, about 1 ⁇ M to about 100 ⁇ M, about 10 ⁇ M to about 1000 ⁇ M, about 10 ⁇ M to about 900 ⁇ M, about 10 ⁇ M to about 800 ⁇ M, about 10 ⁇ M to about 700 ⁇ M, about 10 ⁇ M to about 600 ⁇ M, about 10 ⁇ M to about 500 ⁇ M, about 10 ⁇ M to about 400 ⁇ M, about 10 ⁇ M to about 300 ⁇ M, about 10 ⁇ M to about 200 ⁇ M, about 10 ⁇ M to about 100 ⁇ M, about 100
  • the second detection reagent may include SCS (ADP forming), ADP, a bioluminescent enzyme, a luciferin substrate, and a buffer, such as MOPS, Tris or HEPES.
  • the SCS (ADP forming) enzyme may be a SCS (ADP forming) enzyme from a eukaryote or a prokaryote.
  • the SCS (ADP forming) enzyme may be a SCS (ADP forming) enzyme from Sus scrofa or from E. coli.
  • the SCS (ADP forming) enzyme may be encoded by a succinyl-CoA synthase alpha and beta subunit gene sequences from a eukaryote or a prokaryote.
  • the SCS (ADP forming) enzyme may be encoded by a succinyl-CoA synthase alpha and beta subunit gene sequences from Sus scrofa or from E. coli.
  • the second detection reagent may include from about 0.0001 U/ ⁇ L to about 0.1500 U/ ⁇ L, about 0.0010 U/ ⁇ L to about 0.1500 U/ ⁇ L, about 0.0100 U/ ⁇ L to about 0.1500 U/ ⁇ L, about 0.1000 U/ ⁇ L to about 0.1500 U/ ⁇ L, 0.0001 U/ ⁇ L to about 0.1000 U/ ⁇ L, about 0.0010 U/ ⁇ L to about 0.1000 U/ ⁇ L, about 0.0100 U/ ⁇ L to about 0.1000 U/ ⁇ L, 0.0001 U/ ⁇ L to about 0.0100 U/ ⁇ L, or about 0.0010 U/ ⁇ L to about 0.0100 U/ ⁇ L, or at least about 0.0001 U/ ⁇ L, at least about 0.0002 U/ ⁇ L, at least about 0.0003 U/ ⁇ L, at least about 0.0004 U/ ⁇ L, at least about 0.0005 U/ ⁇ L, at least about 0.0006 U/ ⁇ L, at least about 0.0010 U/ ⁇ L, at least about 0.0013 U/ ⁇ L
  • the second detection reagent may include at least about 0.5 ⁇ M, at least about 1.0 ⁇ M, at least about 1.5 ⁇ M, at least about 2.0 ⁇ M, at least about 2.1 ⁇ M, at least about 2.2 ⁇ M, at least about 2.3 ⁇ M, at least about 2.4 ⁇ M, at least about 2.5 ⁇ M, at least about 2.6 ⁇ M, at least about 2.7 ⁇ M, at least about 2.8 ⁇ M, at least about 2.9 ⁇ M, at least about 3.0 ⁇ M, at least about 3.5 ⁇ M, at least about 4.0 ⁇ M, or at least about 5.0 ⁇ M of ADP.
  • the enzyme SCOT catalyzes the conversion of succinate and acetoacetyl-CoA into acetoacetate and succinyl-CoA (see Fig. 1 ).
  • the intermediate succinyl-CoA in the presence of ADP and inorganic phosphate, is converted into succinate, CoA, and ATP by the enzyme SCS-ADP in a second reaction (see Fig. 1 ).
  • the ATP that is generated is subsequently converted into a bioluminescent signal, e.g., bioluminescence using an ATP detection reagent, e.g., a luciferin-luciferase reaction (See Fig. 16 ).
  • the conversion of succinate to ATP is performed using a reagent that contains SCOT, acetoacetyl-CoA, potassium phosphate (monobasic/dibasic), succinyl-CoA ligase (ADP forming)(SCS-ADP), ADP, magnesium sulfate or other divalent cation (such as magnesium chloride), and a buffer such as MOPS, Tris, or HEPES.
  • a reagent that contains SCOT, acetoacetyl-CoA, potassium phosphate (monobasic/dibasic), succinyl-CoA ligase (ADP forming)(SCS-ADP), ADP, magnesium sulfate or other divalent cation (such as magnesium chloride), and a buffer such as MOPS, Tris, or HEPES.
  • the ATP may then be measured by the addition of an ATP detection reagent, e.g., a luciferin-luciferase assay reagent, e.g., the Kinase-Glo® assay reagent (Promega Corp.).
  • an ATP detection reagent e.g., a luciferin-luciferase assay reagent, e.g., the Kinase-Glo® assay reagent (Promega Corp.).
  • Magnesium is required for SCS-ADP activity as an absence of magnesium causes a 10-fold decrease in activity for this assay.
  • the amount of luminescence generated is proportional to the amount of succinate present in the sample.
  • the first detection reagent may include SCOT, inorganic phosphate, such as potassium phosphate (monobasic/dibasic), acetoacetyl-CoA (also known as 3-oxoacyl-CoA), SCS (ADP forming; SCS-ADP), ADP, magnesium sulfate or other divalent cation (such as magnesium chloride), and a buffer, such as MOPS, Tris, or HEPES.
  • the SCOT enzyme may be a SCOT enzyme from a eukaryote or a prokaryote.
  • the SCOT enzyme may be a SCOT enzyme from Sus scrofa.
  • the SCOT enzyme may be encoded by a 3-oxoacid CoA-transferase gene sequence from a eukaryote or a prokaryote.
  • the SCOT enzyme may be encoded by a 3-oxoacid CoA-transferase gene sequence from Sus scrofa.
  • the first detection reagent may include from about 0.001 ng/ ⁇ L to about 500.00 ng/ ⁇ L, about 0.010 ng/ ⁇ L to about 500.00 ng/ ⁇ L, about 0.10 ng/ ⁇ L to about 500.00 ng/ ⁇ L, about 1.0 ng/ ⁇ L to about 500.00 ng/ ⁇ L, about 10.0 ng/ ⁇ L to about 500.00 ng/ ⁇ L, about 100.00 ng/ ⁇ L to about 500.00 ng/ ⁇ L, about 0.001 ng/ ⁇ L to about 400.00 ng/ ⁇ L, about 0.010 ng/ ⁇ L to about 400.00 ng/ ⁇ L, about 0.10 ng/ ⁇ L to about 400.00 ng/ ⁇ L, about 1.0 ng/ ⁇ L to about 400.00 ng/ ⁇ L, about 10.0 ng/ ⁇ L to about 400.00 ng/ ⁇ L, about 100.00 ng/ ⁇ L to about 400.00 ng/ ⁇ L, about 0.001 ng/ ⁇ L to about 300.00 ng/ ⁇ L, about 0.010 ng
  • the first detection reagent may include from about 50 mM to about 1000 mM, from about 50 mM to about 750 mM, from about 50 mM to about 500 mM, from about 50 mM to about 250 mM, about 100 mM to about 1000 mM, from about 100 mM to about 750 mM, from about 100 mM to about 500 mM, from about 100 mM to about 250 mM, about 250 mM to about 1000 mM, from about 250 mM to about 750 mM, from about 250 mM to about 500 mM, about 500 mM to about 1000 mM, or from about 500 mM to about 750 mM, or at least about 50 mM, at least about 100 mM, at least about 150 mM, at least about 200 mM, at least about 250 mM, at least about 300 mM, at least about 350 mM, at least about 400 mM, at least about 450 mM, at
  • the first detection reagent may include from about 1 ⁇ M to about 1000 ⁇ M, about 1 ⁇ M to about 900 ⁇ M, about 1 ⁇ M to about 800 ⁇ M, about 1 ⁇ M to about 700 ⁇ M, about 1 ⁇ M to about 600 ⁇ M, about 1 ⁇ M to about 500 ⁇ M, about 1 ⁇ M to about 400 ⁇ M, about 1 ⁇ M to about 300 ⁇ M, about 1 ⁇ M to about 200 ⁇ M, about 1 ⁇ M to about 100 ⁇ M, about 10 ⁇ M to about 1000 ⁇ M, about 10 ⁇ M to about 900 ⁇ M, about 10 ⁇ M to about 800 ⁇ M, about 10 ⁇ M to about 700 ⁇ M, about 10 ⁇ M to about 600 ⁇ M, about 10 ⁇ M to about 500 ⁇ M, about 10 ⁇ M to about 400 ⁇ M, about 10 ⁇ M to about 300 ⁇ M, about 10 ⁇ M to about 200 ⁇ M, about 10 ⁇ M to about 100 ⁇ M, about 100
  • the first detection reagent may include from about 0.0001 U/ ⁇ L to about 0.1500 U/ ⁇ L, about 0.0010 U/ ⁇ L to about 0.1500 U/ ⁇ L, about 0.0100 U/ ⁇ L to about 0.1500 U/ ⁇ L, about 0.1000 U/ ⁇ L to about 0.1500 U/ ⁇ L, 0.0001 U/ ⁇ L to about 0.1000 U/ ⁇ L, about 0.0010 U/ ⁇ L to about 0.1000 U/ ⁇ L, about 0.0100 U/ ⁇ L to about 0.1000 U/ ⁇ L, 0.0001 U/ ⁇ L to about 0.0100 U/ ⁇ L, or about 0.0010 U/ ⁇ L to about 0.0100 U/ ⁇ L, or at least about 0.0001 U/ ⁇ L, at least about 0.0002 U/ ⁇ L, at least about 0.0003 U/ ⁇ L, at least about 0.0004 U/ ⁇ L, at least about 0.0005 U/ ⁇ L, at least about 0.0006 U/ ⁇ L, at least about 0.0010 U/ ⁇ L, at least about 0.0013 U/ ⁇ L
  • the first detection reagent may include at least about 0.5 ⁇ M, at least about 1.0 ⁇ M, at least about 1.5 ⁇ M, at least about 2.0 ⁇ M, at least about 2.1 ⁇ M, at least about 2.2 ⁇ M, at least about 2.3 ⁇ M, at least about 2.4 ⁇ M, at least about 2.5 ⁇ M, at least about 2.6 ⁇ M, at least about 2.7 ⁇ M, at least about 2.8 ⁇ M, at least about 2.9 ⁇ M, at least about 3.0 ⁇ M, at least about 3.5 ⁇ M, at least about 4.0 ⁇ M, or at least about 5.0 ⁇ M of ADP.
  • the first detection reagent may include from about 0.05 mM to about 50 mM, about 0.05 mM to about 25 mM, about 0.05 mM to about 12.5 mM, about 0.05 mM to about 6.25 mM, about 0.05 mM to about 3.13 mM, about 0.05 mM to about 1.56 mM, about 0.05 mM to about 0.78 mM, about 0.05 mM to about 0.39 mM, about 0.05 mM to about 0.19 mM, about 0.05 mM to about 0.10 mM, about 0.10 mM to about 50 mM, about 0.10 mM to about 25 mM, about 0.10 mM to about 12.5 mM, about 0.10 mM to about 6.25 mM, about 0.10 mM to about 3.13 mM, about 0.10 mM to about 1.56 mM, about 0.10 mM to about 0.78 mM, about 0.10 mM to about 0.39
  • the second detection reagent may include a bioluminescent enzyme, a luciferin substrate, and a buffer, such as MOPS, Tris, or HEPES.
  • the enzyme GDP-forming succinyl-CoA synthase catalyzes the conversion of succinate, CoA, and GTP to succinyl-CoA, GDP, and Pi.
  • the conversion of succinate to succinyl-CoA is performed using a reagent that contains GDP-forming succinyl-CoA synthase ("SCS-GDP"), GTP, magnesium sulfate or other divalent cation (such as magnesium chloride), and coenzyme A.
  • SCS-GDP GDP-forming succinyl-CoA synthase
  • GTP GTP
  • magnesium sulfate or other divalent cation such as magnesium chloride
  • coenzyme A coenzyme A.
  • the conversion of GDP to ATP may be performed separately using a reagent containing guanylate kinase and ADP.
  • the ATP may then be measured by the addition of an ATP detection reagent, e.g., a luciferin-luciferase assay reagent, e.g., the Kinase-Glo® assay reagent (Promega Corp.).
  • an ATP detection reagent e.g., a luciferin-luciferase assay reagent, e.g., the Kinase-Glo® assay reagent (Promega Corp.).
  • Magnesium is required for both SDS-GDP and GMPK activity in this assay.
  • the amount of luminescence generated is proportional to the amount of succinate present in the sample (see Fig. 8 ).
  • the first detection reagent includes SCS-GDP, GTP, coenzyme A, and a buffer, such as MOPS, Tris or HEPES.
  • the SCS-GDP enzyme may be a SCS-GDP enzyme from a eukaryote or a prokaryote.
  • the SCS-GDP enzyme may be a SCS-GDP enzyme from Sus scrofa.
  • the SCS-GDP enzyme may be encoded by a succinate-CoA ligase, alpha and beta subunit gene sequences from a eukaryote or a prokaryote.
  • the SCS-GDP enzyme may be encoded by a succinate-CoA ligase, alpha and beta subunit gene sequences from Sus scrofa.
  • the first detection reagent may include from about 0.50 ⁇ g/mL to about 100 ⁇ g/mL, about 0.50 ⁇ g/mL to about 50 ⁇ g/mL, about 0.50 ⁇ g/mL to about 25 ⁇ g/mL, about 0.50 ⁇ g/mL to about 10 ⁇ g/mL, about 5.0 ⁇ g/mL to about 100 ⁇ g/mL, about 5.0 ⁇ g/mL to about 50 ⁇ g/mL, about 5.0 ⁇ g/mL to about 25 ⁇ g/mL, about 5.0 ⁇ g/mL to about 10 ⁇ g/mL, about 10.0 ⁇ g/mL to about 100 ⁇ g/mL, about 10.0 ⁇ g/mL to about 50 ⁇ g/mL, or about 10.0 ⁇ g/mL to about 25 ⁇ g/mL, or at least about 0.50 ⁇ g/mL, at least about 0.78 ⁇ g/mL, at least about 1 ⁇ g/mL
  • the first detection reagent may include from about 1 ⁇ M to about 30 ⁇ M, about 1 ⁇ M to about 20 ⁇ M, about 1 ⁇ M to about 10 ⁇ M, about 5 ⁇ M to about 30 ⁇ M, about 5 ⁇ M to about 20 ⁇ M, or about 5 ⁇ M to about 10 ⁇ M, or at least about 1 ⁇ M, at least about 5 ⁇ M, at least about 10 ⁇ M, at least about 11 ⁇ M, at least about 12 ⁇ M, at least about 13 ⁇ M, at least about 14 ⁇ M, at least about 15 ⁇ M, at least about 16 ⁇ M, at least about 17 ⁇ M, at least about 18 ⁇ M, at least about 19 ⁇ M, at least about 20 ⁇ M, at least about 25 ⁇ M, or at least about 30 ⁇ M of GTP.
  • the first detection reagent may include from about 1 ⁇ M to about 25 ⁇ M, about 1 ⁇ M to about 20 ⁇ M, about 1 ⁇ M to about 10 ⁇ M, about 5 ⁇ M to about 25 ⁇ M, about 5 ⁇ M to about 20 ⁇ M, or about 5 ⁇ M to about 10 ⁇ M, or at least about 1 ⁇ M, at least about 5 ⁇ M, at least about 10 ⁇ M, at least about 11 ⁇ M, at least about 12 ⁇ M, at least about 13 ⁇ M, at least about 14 ⁇ M, at least about 15 ⁇ M, at least about 16 ⁇ M, at least about 17 ⁇ M, at least about 110 ⁇ M, at least about 18 ⁇ M, at least about 19 ⁇ M, at least about 20 ⁇ M, or at least about 25 ⁇ M of coenzyme A.
  • the first detection reagent may include from about 0.05 mM to about 50 mM, about 0.05 mM to about 25 mM, about 0.05 mM to about 12.5 mM, about 0.05 mM to about 6.25 mM, about 0.05 mM to about 3.13 mM, about 0.05 mM to about 1.56 mM, about 0.05 mM to about 0.78 mM, about 0.05 mM to about 0.39 mM, about 0.05 mM to about 0.19 mM, about 0.05 mM to about 0.10 mM, about 0.10 mM to about 50 mM, about 0.10 mM to about 25 mM, about 0.10 mM to about 12.5 mM, about 0.10 mM to about 6.25 mM, about 0.10 mM to about 3.13 mM, about 0.10 mM to about 1.56 mM, about 0.10 mM to about 0.78 mM, about 0.10 mM to about 0.39
  • the second detection reagent may include guanylate kinase (GMPK), ADP, a bioluminescent enzyme, a luciferin substrate, and a buffer, such as MOPS, Tris or HEPES.
  • the GMPK enzyme may be a GMPK enzyme from a eukaryote or a prokaryote.
  • the GMPK enzyme may be a GMPK enzyme from Bos taurus.
  • the GMPK enzyme may be encoded by a guanylate kinase 1 gene sequence from a eukaryote or a prokaryote.
  • the GMPK enzyme may be encoded by a guanylate kinase 1 gene sequence from Bos taurus.
  • the second detection reagent may include from about 0.01 ⁇ g/mL to about 300 ⁇ g/mL, about 0.01 ⁇ g/mL to about 200 ⁇ g/mL, about 0.01 ⁇ g/mL to about 100 ⁇ g/mL, about 0.01 ⁇ g/mL to about 50 ⁇ g/mL, about 0.10 ⁇ g/mL to about 300 ⁇ g/mL, about 0.10 ⁇ g/mL to about 200 ⁇ g/mL, about 0.10 ⁇ g/mL to about 100 ⁇ g/mL, about 0.10 ⁇ g/mL to about 50 ⁇ g/mL, about 1.0 ⁇ g/mL to about 300 ⁇ g/mL, about 1.0 ⁇ g/mL to about 200 ⁇ g/mL, about 1.0 ⁇ g/mL to about 100 ⁇ g/mL, about 1.0 ⁇ g/mL to about 50 ⁇ g/mL, about 10 ⁇ g/mL to about 300 ⁇ g/mL
  • the second detection reagent may include from about 0.5 ⁇ M to about 5.0 ⁇ M, about 0.5 ⁇ M to about 4.0 ⁇ M, about 0.5 ⁇ M to about 3.0 ⁇ M, about 0.5 ⁇ M to about 2.0 ⁇ M, about 1.0 ⁇ M to about 5.0 ⁇ M, about 1.0 ⁇ M to about 4.0 ⁇ M, about 1.0 ⁇ M to about 3.0 ⁇ M, about 1.0 ⁇ M to about 2.0 ⁇ M, about 2.0 ⁇ M to about 5.0 ⁇ M, about 2.0 ⁇ M to about 4.0 ⁇ M, about 2.0 ⁇ M to about 3.0 ⁇ M, about 3.0 ⁇ M to about 5.0 ⁇ M, about 3.0 ⁇ M to about 4.0 ⁇ M, or about 4.0 ⁇ M to about 5.0 ⁇ M, or at least about 0.5 ⁇ M, at least about 1.0 ⁇ M, at least about 1.5 ⁇ M, at least about 2.0 ⁇ M, at least about 2.1 ⁇ M, at least about 2.2 ⁇ M,
  • the enzyme GDP-forming succinyl-CoA synthase catalyzes the conversion of succinate, CoA, and GTP to succinyl-CoA, GDP, and Pi.
  • the conversion of succinate to succinyl-CoA is performed using a reagent that contains GDP-forming succinyl-CoA synthase ("SCS-GDP"), GTP, magnesium sulfate or other divalent cation (such as magnesium chloride), and coenzyme A.
  • SCS-GDP GDP-forming succinyl-CoA synthase
  • GTP GTP
  • magnesium sulfate or other divalent cation such as magnesium chloride
  • coenzyme A coenzyme A.
  • the conversion of GDP to ATP may be performed simultaneously using a reagent containing guanylate kinase and ADP.
  • the ATP that is generated may then be measured by the addition of an ATP detection reagent, e.g., a luciferin-luciferase assay reagent, e.g., the Kinase-Glo® assay reagent (Promega Corp.).
  • an ATP detection reagent e.g., a luciferin-luciferase assay reagent, e.g., the Kinase-Glo® assay reagent (Promega Corp.).
  • the amount of luminescence generated is proportional to the amount of succinate present in the sample
  • the first detection reagent includes SCS-GDP, GTP, coenzyme A, guanylate kinase (GMPK), ADP, and a buffer, such as MOPS, Tris or HEPES.
  • the SCS-GDP enzyme may be a SCS-GDP enzyme from a eukaryote or a prokaryote.
  • the SCS-GDP enzyme may be a SCS-GDP enzyme from Sus scrofa.
  • the SCS-GDP enzyme may be encoded by a succinate-CoA ligase, alpha and beta subunit gene sequences from a eukaryote or a prokaryote.
  • the SCS-GDP enzyme may be encoded by a succinate-CoA ligase, alpha and beta subunit gene sequences from Sus scrofa.
  • the GMPK enzyme may be a GMPK enzyme from a eukaryote or a prokaryote.
  • the GMPK enzyme may be a GMPK enzyme from Bos taurus.
  • the GMPK enzyme may be encoded by a guanylate kinase 1 gene sequence from a eukaryote or a prokaryote.
  • the GMPK enzyme may be encoded by a guanylate kinase 1 gene sequence from Bos taurus.
  • the first detection reagent may include from about 0.50 ⁇ g/mL to about 100 ⁇ g/mL, about 0.50 ⁇ g/mL to about 50 ⁇ g/mL, about 0.50 ⁇ g/mL to about 25 ⁇ g/mL, about 0.50 ⁇ g/mL to about 10 ⁇ g/mL, about 5.0 ⁇ g/mL to about 100 ⁇ g/mL, about 5.0 ⁇ g/mL to about 50 ⁇ g/mL, about 5.0 ⁇ g/mL to about 25 ⁇ g/mL, about 5.0 ⁇ g/mL to about 10 ⁇ g/mL, about 10.0 ⁇ g/mL to about 100 ⁇ g/mL, about 10.0 ⁇ g/mL to about 50 ⁇ g/mL, or about 10.0 ⁇ g/mL to about 25 ⁇ g/mL, or at least about 0.50 ⁇ g/mL, at least about 0.78 ⁇ g/mL, at least about 1 ⁇ g/mL
  • the first detection reagent may include from about 1 ⁇ M to about 30 ⁇ M, about 1 ⁇ M to about 20 ⁇ M, about 1 ⁇ M to about 10 ⁇ M, about 5 ⁇ M to about 30 ⁇ M, about 5 ⁇ M to about 20 ⁇ M, or about 5 ⁇ M to about 10 ⁇ M, or at least about 1 ⁇ M, at least about 5 ⁇ M, at least about 10 ⁇ M, at least about 11 ⁇ M, at least about 12 ⁇ M, at least about 13 ⁇ M, at least about 14 ⁇ M, at least about 15 ⁇ M, at least about 16 ⁇ M, at least about 17 ⁇ M, at least about 18 ⁇ M, at least about 19 ⁇ M, at least about 20 ⁇ M, at least about 25 ⁇ M, or at least about 30 ⁇ M of GTP.
  • the first detection reagent may include from about 1 ⁇ M to about 25 ⁇ M, about 1 ⁇ M to about 20 ⁇ M, about 1 ⁇ M to about 10 ⁇ M, about 5 ⁇ M to about 25 ⁇ M, about 5 ⁇ M to about 20 ⁇ M, or about 5 ⁇ M to about 10 ⁇ M, or at least about 1 ⁇ M, at least about 5 ⁇ M, at least about 10 ⁇ M, at least about 11 ⁇ M, at least about 12 ⁇ M, at least about 13 ⁇ M, at least about 14 ⁇ M, at least about 15 ⁇ M, at least about 16 ⁇ M, at least about 17 ⁇ M, at least about 110 ⁇ M, at least about 18 ⁇ M, at least about 19 ⁇ M, at least about 20 ⁇ M, or at least about 25 ⁇ M of coenzyme A.
  • the first detection reagent may include from about 0.01 ⁇ g/mL to about 300 ⁇ g/mL, about 0.01 ⁇ g/mL to about 200 ⁇ g/mL, about 0.01 ⁇ g/mL to about 100 ⁇ g/mL, about 0.01 ⁇ g/mL to about 50 ⁇ g/mL, about 0.10 ⁇ g/mL to about 300 ⁇ g/mL, about 0.10 ⁇ g/mL to about 200 ⁇ g/mL, about 0.10 ⁇ g/mL to about 100 ⁇ g/mL, about 0.10 ⁇ g/mL to about 50 ⁇ g/mL, about 1.0 ⁇ g/mL to about 300 ⁇ g/mL, about 1.0 ⁇ g/mL to about 200 ⁇ g/mL, about 1.0 ⁇ g/mL to about 100 ⁇ g/mL, about 1.0 ⁇ g/mL to about 50 ⁇ g/mL, about 10 ⁇ g/mL to about 300 ⁇ g/mL
  • the first detection reagent may include from about 0.5 ⁇ M to about 5.0 ⁇ M, about 0.5 ⁇ M to about 4.0 ⁇ M, about 0.5 ⁇ M to about 3.0 ⁇ M, about 0.5 ⁇ M to about 2.0 ⁇ M, about 1.0 ⁇ M to about 5.0 ⁇ M, about 1.0 ⁇ M to about 4.0 ⁇ M, about 1.0 ⁇ M to about 3.0 ⁇ M, about 1.0 ⁇ M to about 2.0 ⁇ M, about 2.0 ⁇ M to about 5.0 ⁇ M, about 2.0 ⁇ M to about 4.0 ⁇ M, about 2.0 ⁇ M to about 3.0 ⁇ M, about 3.0 ⁇ M to about 5.0 ⁇ M, about 3.0 ⁇ M to about 4.0 ⁇ M, or about 4.0 ⁇ M to about 5.0 ⁇ M, or at least about 0.5 ⁇ M, at least about 1.0 ⁇ M, at least about 1.5 ⁇ M, at least about 2.0 ⁇ M, at least about 2.1 ⁇ M, at least about 2.2 ⁇ M,
  • the first detection reagent may include from about 0.05 mM to about 50 mM, about 0.05 mM to about 25 mM, about 0.05 mM to about 12.5 mM, about 0.05 mM to about 6.25 mM, about 0.05 mM to about 3.13 mM, about 0.05 mM to about 1.56 mM, about 0.05 mM to about 0.78 mM, about 0.05 mM to about 0.39 mM, about 0.05 mM to about 0.19 mM, about 0.05 mM to about 0.10 mM, about 0.10 mM to about 50 mM, about 0.10 mM to about 25 mM, about 0.10 mM to about 12.5 mM, about 0.10 mM to about 6.25 mM, about 0.10 mM to about 3.13 mM, about 0.10 mM to about 1.56 mM, about 0.10 mM to about 0.78 mM, about 0.10 mM to about 0.39
  • the second detection reagent may include a bioluminescent enzyme, a luciferin substrate, and a buffer, such as MOPS, Tris or HEPES.
  • the enzyme ADP-forming succinyl-CoA synthase catalyzes the conversion of succinate, CoA, and ATP to succinyl-CoA, ADP, and Pi.
  • the conversion of succinate to succinyl-CoA is performed using a reagent that contains SCS-ADP, ATP, magnesium sulfate or other divalent cation (such as magnesium chloride), and coenzyme A.
  • the ATP present or remaining after the conversion of succinate to succinyl-CoA is depleted using a reagent, such as ADP-GloTM Reagent (Promega Corp) containing adenylate cyclase and pyrophosphatase.
  • ADP-GloTM Reagent Promega Corp
  • the conversion of ADP to ATP may be performed separately using a reagent that contains pyruvate kinase and phosphoenolpyruvate.
  • the converted ATP may be measured by luminescence using an ATP detection reagent, e.g., a luciferin-luciferase reagent, e.g., Kinase-Glo (Promega Corp).
  • the conversion of ADP to ATP and the generation of luminescence may be performed simultaneously using a reagent such as ADP-GloTM Kinase Detection Reagent (Promega Corp).
  • the first detection reagent includes SCS-ADP, ATP, coenzyme A, and a buffer, such as MOPS, Tris, or HEPES.
  • the SCS (ADP forming) enzyme may be a SCS (ADP forming) enzyme from a eukaryote or a prokaryote.
  • the SCS (ADP forming) enzyme may be a SCS (ADP forming) enzyme from Sus scrofa or E. coli.
  • the SCS (ADP forming) enzyme may be encoded by a succinyl-CoA synthase alpha and beta subunit gene sequences from a eukaryote or a prokaryote.
  • the SCS (ADP forming) enzyme may be encoded by a succinyl-CoA synthase alpha and beta subunit gene sequences from Sus scrofa or E. coli.
  • the first detection reagent may include from about 0.05 Units/mL to about 1.0 Units/mL, about 0.10 Units/mL to about 1.0 Units/mL, about 0.25 Units/mL to about 1.0 Units/mL, or about 0.50 Units/mL to about 1.0 Units/mL, or at least about 0.05 Units/mL, at least about 0.10 Units/mL, at least about 0.15 Units/mL, at least about 0.20 Units/mL, at least about 0.21 Units/mL, at least about 0.22 Units/mL, at least about 0.23 Units/mL, at least about 0.24 Units/mL, at least about 0.25 Units/mL, at least about 0.26 Units/mL, at least about 0.27 Units/mL, at least about 0.28 Units/mL, at least about 0.29 Units/mL, at least about 0.30 Units/mL, at least about 0.35 Units/mL, at least about 0.40 Unit
  • the first detection reagent may include from about 5 ⁇ M to about 100 ⁇ M, about 5 ⁇ M to about 70 ⁇ M, about 5 ⁇ M to about 50 ⁇ M, about 5 ⁇ M to about 25 ⁇ M, about 10 ⁇ M to about 100 ⁇ M, about 10 ⁇ M to about 70 ⁇ M, about 10 ⁇ M to about 50 ⁇ M, about 10 ⁇ M to about 25 ⁇ M, about 25 ⁇ M to about 100 ⁇ M, about 25 ⁇ M to about 70 ⁇ M, about 25 ⁇ M to about 50 ⁇ M, about 50 ⁇ M to about 100 ⁇ M, or about 50 ⁇ M to about 70 ⁇ M, or at least about 5 ⁇ M, at least about 10 ⁇ M, at least about 15 ⁇ M, at least about 20 ⁇ M, at least about 25 ⁇ M, at least about 26 ⁇ M, at least about 27 ⁇ M, at least about 28 ⁇ M, at least about 29 ⁇ M, at least about 30 ⁇ M, at least about 31 ⁇ M
  • the first detection reagent may include from about 5 ⁇ M to about 100 ⁇ M, about 5 ⁇ M to about 70 ⁇ M, about 5 ⁇ M to about 50 ⁇ M, about 5 ⁇ M to about 25 ⁇ M, about 10 ⁇ M to about 100 ⁇ M, about 10 ⁇ M to about 70 ⁇ M, about 10 ⁇ M to about 50 ⁇ M, about 10 ⁇ M to about 25 ⁇ M, about 25 ⁇ M to about 100 ⁇ M, about 25 ⁇ M to about 70 ⁇ M, about 25 ⁇ M to about 50 ⁇ M, about 50 ⁇ M to about 100 ⁇ M, or about 50 ⁇ M to about 70 ⁇ M, or at least about 5 ⁇ M, at least about 10 ⁇ M, at least about 15 ⁇ M, at least about 20 ⁇ M, at least about 25 ⁇ M, at least about 26 ⁇ M, at least about 27 ⁇ M, at least about 28 ⁇ M, at least about 29 ⁇ M, at least about 30 ⁇ M, at least about 31 ⁇ M
  • the first detection reagent may include from about 0.05 mM to about 50 mM, about 0.05 mM to about 25 mM, about 0.05 mM to about 12.5 mM, about 0.05 mM to about 6.25 mM, about 0.05 mM to about 3.13 mM, about 0.05 mM to about 1.56 mM, about 0.05 mM to about 0.78 mM, about 0.05 mM to about 0.39 mM, about 0.05 mM to about 0.19 mM, about 0.05 mM to about 0.10 mM, about 0.10 mM to about 50 mM, about 0.10 mM to about 25 mM, about 0.10 mM to about 12.5 mM, about 0.10 mM to about 6.25 mM, about 0.10 mM to about 3.13 mM, about 0.10 mM to about 1.56 mM, about 0.10 mM to about 0.78 mM, about 0.10 mM to about 0.39
  • the second detection reagent may include an ATP depletion reagent and an ADP-to-ATP conversion/detection reagent.
  • the ATP depletion reagent may include an enzyme that depletes ATP from the sample, i.e., the enzyme that depletes ATP from the sample may hydrolyze or convert the ATP in the sample.
  • the enzyme may include an adenylate cyclase and ATP sulfurylase.
  • the ATP depletion reagent may include adenylate cyclase, pyrophosphatase, magnesium, calmodulin, and a buffer, such as Tris or HEPES buffer pH 7.5.
  • the adenylate cyclase enzyme may be an adenylate cyclase enzyme from a eukaryote or a prokaryote.
  • the adenylate cyclase enzyme may be an adenylate cyclase enzyme from Bordetella pertussis toxin.
  • the adenylate cyclase enzyme may be encoded by an adenylate cyclase gene sequence from a eukaryote or a prokaryote.
  • the adenylate cyclase enzyme may be encoded by an adenylate cyclase gene sequence from Bordetella pertussis toxin.
  • the adenylate cyclase enzyme may be encoded by an adenylate cyclase gene sequence from Bordetella pertussis toxin.
  • the ATP depletion reagent may include from about 100 ⁇ g/mL to about 200 ⁇ g/mL, about 125 ⁇ g/mL to about 200 ⁇ g/mL, about 150 ⁇ g/mL to about 200 ⁇ g/mL, about 175 ⁇ g/mL to about 200 ⁇ g/mL, about 100 ⁇ g/mL to about 175 ⁇ g/mL, about 125 ⁇ g/mL to about 175 ⁇ g/mL, about 150 ⁇ g/mL to about 175 ⁇ g/mL, about 100 ⁇ g/mL to about 150 ⁇ g/mL, about 125 ⁇ g/mL to about 200 ⁇ g/mL, about 125 ⁇ g/mL to about 175 ⁇ g/mL, or about 125 ⁇ g/
  • the ATP depletion reagent may be added separately before the ADP-to-ATP conversion/detection reagent.
  • the ATP depletion reaction may be carried out at a temperature for a sufficient time to ensure the complete depletion of ATP, i.e., the complete conversion of all ATP to cAMP.
  • the ADP-to-ATP conversion/detection reagent may be added to simultaneously convert ADP to generate ATP and generate luminescence using the generated ATP.
  • the ADP-to-ATP conversion/detection reagent may include pyruvate kinase (PK), phosphoenolpyruvate, magnesium chloride or other divalent cations, a bioluminescent enzyme, a luciferin substrate, and a buffer, such as MOPS, Tris or HEPES.
  • PK pyruvate kinase
  • phosphoenolpyruvate magnesium chloride or other divalent cations
  • a bioluminescent enzyme such as MOPS, Tris or HEPES.
  • the ATP-to-ATP conversion/detection reagent may be included in the second reagent.
  • the ATP-to-ATP conversion/detection reagent may be in a third reagent
  • Luciferase enzymes produce catalytic products that provide a detectable light product, sensitivity, and allow easy measurement of ATP.
  • any bioluminescence generating-enzyme that is ATP-dependent may be used in the methods and compositions of the present disclosure.
  • luciferases are defined by their ability to produce luminescence. More specifically, a luciferase is an enzyme that catalyzes the oxidation of a luciferin substrate to produce oxyluciferin and photons.
  • beetle luciferases such as that of the common firefly (family Lampyridae), form a distinct class with unique evolutionary origins.
  • Beetle luciferases are often referred to as firefly luciferases in the literature; however, firefly luciferases are actually a subgroup of the beetle luciferase class.
  • Beetle luciferases may be purified from the lanterns of the beetles themselves or from protein expression systems well known in the art.
  • Beetle luciferases particularly firefly luciferase from the North American firefly Photinus pyralis , are well known in the art.
  • the P. pyralis luciferase (LucPpy) consists of approximately 550 amino acids of M r 61 kDa as calculated by the protein encoded by the nucleotide sequence of the gene.
  • other firefly luciferases are known, such as Photuris pennsylvanica firefly luciferase (LucPpe2; 545 amino acid residues; GenBank 2190534).
  • Thermostable and/or chemostable mutant luciferases derived from LucPpe2 e.g., LucPpe2m78 (also known as 78-0B10); LucPpe2m90 (also known as 90-1B5); LucPpe2m133 (also known as 133-1B2); LucPpe2m146 (also known as 146-1H2) may be employed, however, any luciferase that meets the limitations set forth herein may be used in the composition, method and kits of the disclosure.
  • the method of making mutant luciferases from LucPpe is disclosed inWO 01/20002 A1.
  • Luciferases that may be used in the methods, compositions and kits described herein include those found in WO 1999/14336 , WO 2001/20002 , EP 1 124 944 , EP 1 224 294 , U.S. Pat. Nos. 6,171,808 , 6,132,983 , and 6,265,177 .
  • Luciferases can be isolated from biological specimens that produce luciferase or from a cell that expresses an exogenous polynucleotide encoding a desired luciferase. Such techniques are well known to those of skill in the art (see U.S. Pat. No. 6,602,677 ).
  • the naturally-occurring substrate for beetle luciferases is firefly luciferin, a polytherocyclic organic acid, D-(-)-2-(6'-hydroxy-2'-benzoth-iazolyl)- ⁇ 2 -thiazolin-4-carboxylic acid (D-luciferin).
  • Luciferin may be isolated from nature (e.g., from fireflies) or synthesized. Synthetic luciferin can have the same structure as the naturally occurring luciferin or can be derivatized, so long as it functions analogously.
  • derivatives of luciferin include D-luciferin methyl ester and other esters of luciferin that are hydrolyzed or acted upon by esterases in a sample to yield luciferin, and naphthyl- and quinolyl-luciferin (Branchini et al., 1989).
  • luciferin e.g., Promega Corp. Madison, Wis.
  • the beetle luciferase-catalyzed reaction that yields luminescence involves firefly luciferin, adenosine triphosphate (ATP), magnesium, and molecular oxygen.
  • the firefly luciferin and ATP react to form luciferyl adenylate with the elimination of inorganic pyrophosphate.
  • the luciferyl adenylate remains tightly bound to the catalytic site of luciferase.
  • this form of the enzyme is exposed to molecular oxygen, the enzyme-bound luciferyl adenylate is oxidized to yield oxyluciferin in an electronically excited state. The excited oxidized luciferin emits light on returning to the ground state.
  • the present disclosure provides methods for detecting and quantifying succinate.
  • the methods involve contacting the sample with a first detection reagent to form a first reaction mixture, contacting the first reaction mixture with a second detection reagent to form a second reaction mixture, and detecting luminescence in the second reaction mixture, thereby detecting or determining the presence or amount of succinate in the sample.
  • the first detection reagent comprises 3-oxoacid CoA-transferase (SCOT), inorganic phosphate, and acetoacetyl-CoA and the second detection reagent comprises succinyl-CoA ligase (SCS) (e.g., SCS-ADP), ADP, a bioluminescent enzyme, and a luciferin substrate.
  • SCS succinyl-CoA ligase
  • the first detection reagent comprises 3-oxoacid CoA-transferase (SCOT), inorganic phosphate, acetoacetyl-CoA, succinyl-CoA ligase (SCS) (e.g., SCS-ADP), ADP, magnesium chloride, and the second detection reagent comprises a bioluminescent enzyme and a luciferin substrate.
  • the first detection reagent comprises SCS-GDP, GTP, and coenzyme A
  • the second detection reagent comprises guanylate kinase (GMPK), ADP, a bioluminescent enzyme, and a luciferin substrate.
  • the first detection reagent comprises SCS-GDP, GTP, coenzyme A, guanylate kinase (GMPK), and ADP
  • the second detection reagent comprises a bioluminescent enzyme, and a luciferin substrate.
  • the first detection reagent comprises ADP forming succinyl-CoA ligase (SCS-ADP), ATP, and coenzyme A
  • the second detection reagent comprises an ATP depletion reagent and an ADP-to-ATP conversion/detection reagent
  • the first reaction mixture is contacted sequentially with the ATP depletion reagent and subsequently the ADP-to-ATP conversion/detection reagent
  • the ATP depletion reagent comprises adenylate cyclase and pyrophosphatase
  • the ADP-to-ATP conversion/detection reagent comprises pyruvate kinase, phosphoenolpyruvate, a bioluminescent enzyme, and a luciferin substrate.
  • Exemplary ATP depletion reagents and/or ADP-to-ATP conversion/detection reagents are described in U.S. Patent No. 8,183,007 .
  • Any one of the reactions involving any of the enzymes may be restricted or limited by time, enzyme concentration, and/or substrate concentration. Reaction conditions may be adjusted so that the reaction is carried out under conditions that result in about, at least about, or at most about 1, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, 99 or 100% completion, or any range derivable therein.
  • any of the reactions described above may be carried out at a temperature between about 15°C and about 45°C, about 15°C and about 40°C, about 15°C and about 35°C, about 15°C and about 30°C, about 15°C and about 25°C, about 15°C and about 20°C, about 20°C and about 45°C, about 20°C and about 40°C, about 20°C and about 35°C, about 20°C and about 30°C, about 20°C and about 25°C, between about 25°C and about 45°C, about 25°C and about 40°C, about 25°C and about 35°C, about 25°C and about 30°C, between about 30°C and about 45°C, about 30°C and about 40°C, about 30°C and about 35°C, between about 35°C and about 45°C, or between about 35°C and about 40°C.
  • the reactions described above may be carried out at 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, or 45°C.
  • succinyl-CoA by SCOT, SCS-ADP, and/or SCS-GDP may be carried out at a temperature between about 15°C and about 45°C, about 15°C and about 40°C, about 15°C and about 35°C, about 15°C and about 30°C, about 15°C and about 25°C, about 15°C and about 20°C, about 20°C and about 45°C, about 20°C and about 40°C, about 20°C and about 35°C, about 20°C and about 30°C, about 20°C and about 25°C, between about 25°C and about 45°C, about 25°C and about 40°C, about 25°C and about 35°C, about 25°C and about 30°C, between about 30°C and about 45°C, about 30°C and about 40°C, about 30°C and about 35°C, between about 35°C and about 45°C, or between about 35°C and about 40°C.
  • succinyl-CoA by SCOT, SCS-ADP, and/or SCS-GDP may be carried out at 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, or 45°C.
  • the conversion of GDP to ATP by guanylate kinase may be carried out at a temperature between about 15°C and about 45°C, about 15°C and about 40°C, about 15°C and about 35°C, about 15°C and about 30°C, about 15°C and about 25°C, about 15°C and about 20°C, about 20°C and about 45°C, about 20°C and about 40°C, about 20°C and about 35°C, about 20°C and about 30°C, about 20°C and about 25°C, between about 25°C and about 45°C, about 25°C and about 40°C, about 25°C and about 35°C, about 25°C and about 30°C, between about 30°C and about 45°C, about 30°C and about 40°C, about 30°C and about 35°C, between about 35°C and about 45°C, or between about 35°C and about 40°C.
  • the conversion of GDP to ATP by guanylate kinase may be carried out at 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, or 45°C.
  • the depletion of ATP by adenylate cyclase and pyrophosphatase may be carried out at a temperature between about 15°C and about 45°C, about 15°C and about 40°C, about 15°C and about 35°C, about 15°C and about 30°C, about 15°C and about 25°C, about 15°C and about 20°C, about 20°C and about 45°C, about 20°C and about 40°C, about 20°C and about 35°C, about 20°C and about 30°C, about 20°C and about 25°C, between about 25°C and about 45°C, about 25°C and about 40°C, about 25°C and about 35°C, about 25°C and about 30°C, between about 30°C and about 45°C, about 30°C and about 40°C, about 30°C and about 35°C, between about 35°C and about 45°C, or between about 35°C and about 40°C.
  • the depletion of ATP by adenylate cyclase may be carried out at 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, or 45°C.
  • the conversion of ADP to ATP by pyruvate kinase may be carried out at a temperature between about 15°C and about 45°C, about 15°C and about 40°C, about 15°C and about 35°C, about 15°C and about 30°C, about 15°C and about 25°C, about 15°C and about 20°C, about 20°C and about 45°C, about 20°C and about 40°C, about 20°C and about 35°C, about 20°C and about 30°C, about 20°C and about 25°C, between about 25°C and about 45°C, about 25°C and about 40°C, about 25°C and about 35°C, about 25°C and about 30°C, between about 30°C and about 45°C, about 30°C and about 40°C, about 30°C and about 35°C, between about 35°C and about 45°C, or between about 35°C and about 40°C.
  • the conversion of ADP to ATP by pyruvate kinase and phosphoenolpyruvate may be carried out at 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, or 45°C.
  • the amount of ATP generated is determined using a luciferase/luciferin reaction.
  • the ATP is utilized by the luciferase, along with luciferin and sufficient molecular oxygen (O 2 ) to drive the detection of ATP, thereby generating AMP, PPi, oxyluciferin, CO 2 , and light.
  • the herein described assay generates measurable luminescence. Quantifying the amount of luminescence quantifies the amount of ATP, and thus the amount of succinate in a sample.
  • the luminescence (relative light units; RLUs) measured is proportional to the level of succinate present in the sample.
  • Quantitative ATP values are realized, for example, when the luminescence generated from a test sample, is compared to the luminescence generated from a control sample or to a standard curve determined by using known amounts of ATP and/or succinate, and the same luciferase and reaction conditions (i.e., temperature, pH, etc.). It is understood that quantification involves subtraction of background values or calculating the signal above background.
  • Qualitative ATP values are realized when the luminescence generated from one sample is compared to the luminescence generated from another sample without a need to know the absolute amount of succinate present in the samples. Alternatively, the sample may be depleted of any intrinsic ATP that could interfere with the read out of the detection of ATP.
  • the herein described method may involve comparing the luminescence results to a control or a comparative sample.
  • the control or comparative sample may contain a known amount of succinate.
  • a standard curve of succinate using a series of calibrating compositions may be performed to correlate the luminescence (RLUs) with the amount of succinate present in the sample.
  • the present disclosure also provides methods for detecting and quantifying succinate forming or generating enzymes and/or their activities.
  • the method generates measurable luminescence, which is proportional to the level of succinate that is formed by the succinate forming or generating enzymes in a succinate forming or generating reaction, such as a demethylation reaction, in the sample.
  • the succinate forming or generating reaction may involve contacting the sample, which contains the succinate forming or generating enzyme, with a peptide or protein substrate, such as a histone or peptide substrate, 2-oxoglutarate, Fe(II), and ascorbate to form a succinate reaction mixture.
  • the succinate reaction mixture is then contacted with the first detection reagent and the second detection reagent, as described above.
  • Succinate is formed in the first reaction mixture, i.e., the succinate forming or generating reaction, if the sample contains a succinate forming or generating enzyme, such as 2-oxoglutarate oxygenase, such as a JMJC demethylase or 2OG-dependent dioxygenase.
  • a succinate forming or generating enzyme such as 2-oxoglutarate oxygenase, such as a JMJC demethylase or 2OG-dependent dioxygenase.
  • 2-oxoglutarate oxygenases catalyze two electron oxidation reactions by coupling the oxidation of a substrate to the oxidative decarboxylation of 2OG, giving succinate and carbon dioxide co-products.
  • the succinate that is formed in the succinate reaction mixture is converted to succinyl-CoA in the first reaction mixture. Changes in succinate are used to determine the activity of the 2-oxoglutarate oxygenase, such as the activity of JMJC demethylases and/or 2OG-dependent dioxygenase.
  • the succinyl-CoA is converted to ATP, and the amount of ATP generated is determined using a luciferase/luciferin reaction, and the light produced is proportional to the succinate forming or generating enzyme or succinate forming or generating enzyme activity present in the sample.
  • the methods for detecting and quantifying succinate forming or generating enzymes and/or their activities involve contacting the sample with a peptide, protein, or non-protein substrate, 2-oxoglutarate, Fe(II), and ascorbate to form a succinate reaction mixture, wherein succinate is formed if the sample comprises a succinate forming or generating enzyme; contacting the succinate reaction mixture with a first detection reagent to form a first reaction mixture, and wherein the succinate formed in the previous step is converted to succinyl-CoA; contacting the first reaction mixture with a second detection reagent to form a second reaction mixture, and detecting luminescence in the second reaction mixture, thereby detecting or determining the presence or amount of succinate forming or generating enzyme or succinate forming or generating enzyme activity in the sample.
  • the succinate formed in the previous step is converted to ATP.
  • the first detection reagent comprises 3-oxoacid CoA-transferase (SCOT), inorganic phosphate, and acetoacetyl-CoA and the second detection reagent comprises succinyl-CoA ligase (SCS), e.g., SCS-ADP, ADP, a bioluminescent enzyme, and a luciferin substrate.
  • SCS succinyl-CoA ligase
  • the first detection reagent comprises 3-oxoacid CoA-transferase (SCOT), inorganic phosphate, acetoacetyl-CoA, succinyl-CoA ligase (SCS) (e.g., SCS-ADP), ADP, magnesium chloride, and the second detection reagent comprises a bioluminescent enzyme and a luciferin substrate.
  • the first detection reagent comprises SCS-GDP, GTP, and coenzyme A
  • the second detection reagent comprises guanylate kinase (GMPK), ADP, a bioluminescent enzyme, and a luciferin substrate.
  • the first detection reagent comprises SCS-GDP, GTP, coenzyme A, guanylate kinase (GMPK), and ADP
  • the second detection reagent comprises a bioluminescent enzyme, and a luciferin substrate.
  • the first detection reagent comprises ADP forming succinyl-CoA ligase (SCS-ADP), ATP, and coenzyme A
  • the second detection reagent comprises an ATP depletion reagent
  • a third detection reagent comprising an ADP-to-ATP conversion/detection reagent; wherein the first reaction mixture is contacted sequentially with the ATP depletion reagent and the ADP-to-ATP conversion/detection reagent, and wherein the ATP depletion reagent comprises adenylate cyclase and pyrophosphatase
  • the ADP-to-ATP conversion/detection reagent comprises pyruvate kinase, phosphoenolpyruvate, a bioluminescent enzyme, and a luciferin substrate.
  • the 2-oxoglutarate oxygenase may be a Fe(II)-dependent lysine demethylase, such as a JMJC de
  • Quantifying the amount of luminescence quantifies the amount of ATP, and thus the amount of succinate produced by the 2-oxoglutarate oxygenase in a sample.
  • the amount of succinate produced is directly proportional to the 2-oxoglutarate oxygenase and/or 2-oxoglutarate oxygenase activity.
  • quantitation of ATP allows for quantitation of 2-oxoglutarate oxygenase and/or 2-oxoglutarate oxygenase activity.
  • Quantitative ATP values are realized when the luminescence generated from a test sample, in which succinate is generated by the 2-oxoglutarate oxygenase present in the sample, is compared to the luminescence generated from a control sample or to a standard curve determined by using known amounts of succinate and/or 2-oxoglutarate oxygenase and the same luciferase and reaction conditions (i.e., temperature, pH, etc.). It is understood that quantification involves subtraction of background values or calculating the signal above background.
  • Qualitative ATP values are realized when the luminescence generated from one sample is compared to the luminescence generated from another sample without a need to know the absolute amount of 2-oxoglutarate oxygenase present in the samples.
  • the herein described method may involve comparing the luminescence results to a control or a comparative sample.
  • the control or comparative sample may contain a known amount of succinate and/or 2-oxoglutarate oxygenase.
  • a standard curve of succinate and/or 2-oxoglutarate oxygenase may be generated to correlate the luminescence (RLUs) with the amount of 2-oxoglutarate oxygenase or 2-oxoglutarate oxygenase activity present in the sample.
  • Any one of the reactions involving any of the enzymes may be restricted or limited by time, enzyme concentration, and/or substrate concentration. Reaction conditions may be adjusted so that the reaction is carried out under conditions that result in about, at least about, or at most about 1, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, 99 or 100% completion, or any range derivable therein.
  • the formation of succinate by a 2-oxoglutarate oxygenase may be carried out at between about 15°C and about 45°C, about 15°C and about 40°C, about 15°C and about 35°C, about 15°C and about 30°C, about 15°C and about 25°C, about 15°C and about 20°C, about 20°C and about 45°C, about 20°C and about 40°C, about 20°C and about 35°C, about 20°C and about 30°C, about 20°C and about 25°C, between about 25°C and about 45°C, about 25°C and about 40°C, about 25°C and about 35°C, about 25°C and about 30°C, between about 30°C and about 45°C, about 30°C and about 40°C, about 30°C and about 35°C, between about 35°C and about 45°C, or between about 35°C and about 40°C
  • the formation of succinate by a 2-oxoglutarate oxygenase may be carried out at 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, or 45°C.
  • a 2-oxoglutarate oxygenase such as JMJC demethylase or 2OG-dependent dioxygenase
  • These temperature conditions may be maintained for 1 min, 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 60 min, 65 min, 70 min, 75 min, 80 min, 85 min, 90 min, or more.
  • the 2-oxoglutarate oxygenase may be a Fe(II)-dependent lysine demethylase, such as a histone demethylase.
  • Fe(II)-dependent lysine demethylases may be a JMJC demethylase.
  • a JMJC demethylase is a histone demethylase containing a JumonjiC (JmjC) domain.
  • the JMJC demethylase may be a member of the KDM3, KDM4, KDM5, or KDM6 family of histone demethylases.
  • the JMJC demethylase may be JMJD1A, JMJD1B, JMJD1C, JMJD2A, JMJD2B, JMJD2C, JMJD2D, JMJD3, JMJD4, JMJD5, JMJD6, JARID1A, JARID1B, JARID1C, JARID1D, UTX, or FBXL11.
  • KDM3A also referred to as JHDM2A/JMJD1A/TSGA
  • KDM3A can act on mono- and dimethylated H3K9.
  • the KDM3A has roles in spermatogenesis and metabolic functions. Knockdown studies of KDM3A in mice, where the mouse produces reduced levels of KDM3A, resulted in male infertility and adult onset-obesity. Additional studies have indicated that KDM3A may play a role in regulation of androgen receptor-dependent genes as well as genes involved in pluripotency, indicating a potential role for KDM3A in tumorigenesis.
  • the KDM4 family includes KDM4A, KDM4B, KDM4C, and KDM4D, which are also referred to as JMDM3A/JMJD2A, JMDM3B/JMJD2B, JMDM3C/JMJD2C, and JMDM3D/JMJD2D, respectively. These enzymes can act on di- and trimethylated H3K9, H3K36, H1K26. KDM4B and KDM4C have roles in tumorigenesis. The KDM4 family of proteins has been linked to malignant transformation.
  • KDM4C amplification has been documented in oesophageal squamous carcinomas, medulloblastomas and breast cancers; amplification of KDM4B has also been found in medulloblastomas.
  • Other gene expression data has also suggested KDM4A, KDM4B, and KDM4C are over-expressed in prostate cancer.
  • JMJD2C lysine-specific demethylase 4C
  • This gene is a member of the Jumonji domain 2 (JMJD2) family and encodes a protein with one JmjC domain, one JumonjiN (JmjN) domain, two PHD-type zinc fingers, and two6.1 domains.
  • This nuclear protein functions as a trimethylation-specific demethylase, converting specific trimethylated histone residues to the dimethylated form. Chromosomal aberrations and increased transcriptional expression of this gene are associated with esophageal squamous cell carcinoma.
  • JMJD2A lysine-specific demethylase 4A
  • This gene is a member of the Jumonji domain 2 (JMJD2) family and encodes a protein with a JmjN domain, a JmjC domain, a JD2H domain, two TUDOR domains, and two PHD-type zinc fingers.
  • JMJD2A Jumonji domain 2
  • This nuclear protein functions as a trimethylation-specific demethylase, converting specific trimethylated histone residues to the dimethylated form, and as a transcriptional repressor.
  • the KDM5 family includes KDM5A, KDM5B, KDM5C, and KDM5D, which are also referred to as JARID1A/RBP2, JARID1B/PLU-1, JARID1C/SMCX, and JARID1D/SMCY, respectively. These enzymes can act on di- and trimethylated H3K4.
  • the KDM5 protein family may have developmental functions. KDM5A in cell culture systems have been linked to regulation of differentiation, mitochondrial function, cell cycle progression. KDM5B and KDM5C may interact with PcG proteins, which are involved in transcriptional repression.
  • JARID1A lysine-specific demethylase 5A
  • the protein is a ubiquitously expressed nuclear protein and binds directly, with several other proteins, to retinoblastoma protein which regulates cell proliferation. It was formally known as Retinoblastoma Binding Protein 2 (RBP2) and also interacts with rhombotin-2 which functions distinctly in erythropoiesis and in T-cell leukemogenesis. Rhombotin-2 is thought to either directly affect the activity of the encoded protein or may indirectly modulate the functions of the retinoblastoma protein by binding to this protein. Alternatively spliced transcript variants encoding distinct isoforms have been found for this gene.
  • KDM6A also referred to as UTX
  • KDM6B also referred to as JMJD3
  • Both KDM6A and KDM6B possess tumor-suppressive characteristics.
  • KDM6A knockdowns in fibroblasts lead to an immediate increase in fibroblast population, while KDM6B expressed in fibroblasts induces oncogenes of the RAS_RAF pathway.
  • Deletions and point mutations of KDM6A have been identified as one cause of Kabuki Syndrome, a congenital disorder resulting in intellectual disability. Mutating homologs of KDM6B disrupted gonadal development in C .
  • KDM6B expression is up-regulated in activated macrophages and dynamically expressed during differentiation of stem cells.
  • Depletion of homologs of KDM6A in D. rerio have shown decreased expression of HOX genes, which play a role in regulating body patterning during development.
  • KDM6A has been shown to regulate HOX genes as well.
  • the 2-oxoglutarate oxygenase may be a 2-oxoglutarate (2OG)-dependent dioxygenase.
  • 2OG-dependent dioxygenases ferrous iron (Fe(II)) is coordinated by a (His)2(Glu/Asp)1 "facial triad" motif. Bidentate coordination of 2OG and water completes a pseudo-octahedral coordination sphere. Following substrate binding, the water ligand is released, yielding an open coordination site for oxygen activation.
  • a transformation occurs during which 2OG is oxidatively decarboxylated to succinate and the O-O bond is cleaved to form a Fe(IV)-oxo (ferryl) intermediate.
  • the 2OG-dependent dioxygenases includes enzymes involved with protein modification (e.g., prolyl 4-hydroxylase, prolyl 3-hydroxylase, lysyl hydroxylase, aspartyl-aspariginyl ⁇ -hydroxylase), ribosomal protein hydroxylation (e.g., ribosomal oxygenases YcfD, MINA53 and nucleolar protein 66), oxygen sensing (e.g., PHDs (such as Hypoxia-inducible factor prolyl hydroxylase 2 (also known as"HIF-PH2", “EGLN1", and “PHD2”) and Egl nine homolog 2 (EGLN2)), HIF ⁇ -specific prolyl 4-hydroxylase, factor-inhibiting HIF (FIH)), DNA and RNA demethylation (e.g., AlkB, ALKBH2/3, FTO), DNA and tRNA hydroxylation (
  • TET1 Ten-eleven translocation methylcytosine dioxygenase 1
  • TET1 catalyzes the conversion of the modified DNA base 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC) coupled with the oxidation of alpha-ketoglutarate (the co-substrate) into succinate and CO 2 .
  • TET1 produces 5-hmC by oxidation of 5-mC in an iron and alpha-ketoglutarate dependent manner.
  • the conversion of 5-mC to 5-hmC may be the initial step of active DNA demethylation in mammals.
  • the 2-oxoglutarate oxygenase may be a hydroxylating enzymes involved with HIF-1 ⁇ hydroxylation.
  • Hypoxia-inducible factors are transcription factors that respond to changes in available oxygen in the cellular environment, to be specific, to decreases in oxygen, or hypoxia.
  • HIF-1 ⁇ is a subunit of a heterodimeric transcription factor hypoxia-inducible factor 1 (HIF-1) that is encoded by the HIF1A gene. It is a basic helix-loop-helix PAS domain containing protein, and is considered as the master transcriptional regulator of cellular and developmental response to hypoxia.
  • the dysregulation and overexpression of HIF-1 ⁇ by either hypoxia or genetic alternations have been heavily implicated in cancer biology, as well as a number of other pathophysiologies, specifically in areas of vascularization and angiogenesis, energy metabolism, cell survival, and tumor invasion.
  • the hydroxylating enzyme may be Hypoxia-inducible factor prolyl hydroxylase 2 (also known as"HIF-PH2", “EGLN1", and “PHD2”) or Egl nine homolog 2 (EGLN2)).
  • the peptide or protein substrate is a substrate that is used by the succinate forming or generating enzyme.
  • the peptide or protein substrate may be a substrate for a histone demethylase, such as a Fe(II)-dependent lysine demethylase or a protein hydroxylase.
  • a code has been developed to indicate the substrate for a particular histone demethylase.
  • the substrate is first specified by the histone subunit (H1, H2A, H2B, H3, H4) and then the one letter designation and number of the amino acid that is methylated.
  • the level of methylation is sometimes noted by the addition of "me#", with the numbers being 1, 2, and 3 for monomethylated, dimethylated, and trimethylated substrates, respectively.
  • H3K9me2 is histone H3 with a dimethylated lysine in the ninth position.
  • the peptide or protein substrate may contain a methylation group that is removed during the formation of succinate.
  • the peptide or protein substrate may be histone H3 Me3K9, histone H3 Me2K9, histone H3 Me1K9, histone H3 Me3K4, histone H3 Me2K4, histone H3 Me3K27, histone H3 Me2K27, histone H3 Me3K36, or histone H3 Me2K36.
  • the non-protein substrate is a substrate that is used by the succinate forming or generating enzyme.
  • the non-protein substrate may be a substrate for a 2OG-dependent dioxygenase.
  • the 2OG-dependent dioxygenases may be a Fe(II)-dependent RNA demethylase, a DNA hydroxylase, a synthase, a hydroxylase, or an oxidase involved with antibiotic biosynthesis or biosynthesis of flavonoids, gibberellins, alkaloids and lipid metabolism.
  • the non-protein substrate may contain a methylation group that is removed during the formation of succinate.
  • non-protein substrates include oligonucleotides containing cytosine modifications in DNA or RNA sequences (1- and 3-alkyl groups), DNA methylation and hydroxymethylation (5mC, 5hmC, 5fC and 5caC), tRNA (5-methoxycarbonylmethyluridine), N ⁇ trimethyllysine, phytanoyl-CoA, N- ⁇ -acetyl-L-arginine, deoxyamidinoproclavaminate, deoxyguanidinoproclavaminate, dihydroclavaminate, proclavamic acid, 3'-methylcephem, 3-exomethylenecephalosporin, 7-aminodeacetoxycephalosporanic acid, cephalexin, deacetoxycephalosporin C, phenylacetyl-7-aminodeacetoxycephalosporanic acid, ampicillin, penicillin G, penicillin N, (3R,5R)-carbapenam-3-carboxylate, (3
  • Another aspect of the present disclosure provides for methods for detecting or determining the presence or amount of 2-oxoglutarate oxygenase and/or 2-oxoglutarate oxygenase activity in a sample.
  • the method includes contacting the sample with a peptide, protein, or non-protein substrate, 2-oxoglutarate, Fe(II), and ascorbate to form a succinate reaction mixture, wherein succinate is formed if the sample comprises 2-oxoglutarate oxygenase; the first detection reagent may be added to the 2OG-dependent oxygenase activity assay in order to prevent succinate inhibition of 2OG-dependent oxygenase; contacting the succinate reaction mixture with a first detection reagent to form a first reaction mixture, wherein the succinate formed in step (a) is converted to succinyl-CoA or ATP; contacting the first reaction mixture with a second detection reagent to form a second reaction mixture; and detecting luminescence in the second reaction mixture, thereby detecting or determining the
  • the first detection reagent comprises 3-oxoacid CoA-transferase (SCOT), inorganic phosphate, and acetoacetyl-CoA and the second detection reagent comprises succinyl-CoA ligase (SCS), e.g., SCS-ADP, ADP, a bioluminescent enzyme, and a luciferin substrate.
  • SCS succinyl-CoA ligase
  • the first detection reagent comprises 3-oxoacid CoA-transferase (SCOT), inorganic phosphate, acetoacetyl-CoA, succinyl-CoA ligase (SCS) (e.g., SCS-ADP), ADP, magnesium chloride, and the second detection reagent comprises a bioluminescent enzyme and a luciferin substrate.
  • the first detection reagent comprises SCS-GDP, GTP, and coenzyme A
  • the second detection reagent comprises guanylate kinase (GMPK), ADP, a bioluminescent enzyme, and a luciferin substrate.
  • the first detection reagent comprises SCS-GDP, GTP, coenzyme A, guanylate kinase (GMPK), and ADP
  • the second detection reagent comprises a bioluminescent enzyme, and a luciferin substrate.
  • the first detection reagent comprises ADP forming succinyl-CoA ligase (SCS-ADP), ATP, and coenzyme A
  • the second detection reagent comprises an ATP depletion reagent.
  • a third detection reagent comprises an ADP-to-ATP conversion/detection reagent; wherein the first reaction mixture is contacted sequentially with the ATP depletion reagent and the ADP-to-ATP conversion/detection reagent, and wherein the ATP depletion reagent comprises adenylate cyclase and pyrophosphatase and the ADP-to-ATP conversion/detection reagent comprises pyruvate kinase, phosphoenolpyruvate, a bioluminescent enzyme, and a luciferin substrate.
  • the method includes contacting the sample with a peptide or protein substrate, 2-oxoglutarate, Fe(II), and ascorbate to form a succinate reaction mixture, wherein succinate is formed if the sample comprises JMJC demethylase; the first detection reagent may be added to the JMJC demethylase activity assay in order to prevent succinate inhibition of JMJC demethylase; contacting the succinate reaction mixture with a first detection reagent to form a first reaction mixture, wherein the succinate formed in step (a) is converted to succinyl-CoA or ATP; contacting the first reaction mixture with a second detection reagent to form a second reaction mixture; and detecting luminescence in the second reaction mixture, thereby detecting or determining the presence or amount of JMJC demethylase or JMJC de
  • the first detection reagent comprises 3-oxoacid CoA-transferase (SCOT), inorganic phosphate, and acetoacetyl-CoA
  • the second detection reagent comprises succinyl-CoA ligase (SCS), e.g., SCS-ADP, ADP, a bioluminescent enzyme, and a luciferin substrate.
  • SCS succinyl-CoA ligase
  • the first detection reagent comprises 3-oxoacid CoA-transferase (SCOT), inorganic phosphate, acetoacetyl-CoA, succinyl-CoA ligase (SCS) (e.g., SCS-ADP), ADP, magnesium chloride, and the second detection reagent comprises a bioluminescent enzyme and a luciferin substrate.
  • the first detection reagent comprises SCS-GDP, GTP, and coenzyme A
  • the second detection reagent comprises guanylate kinase (GMPK), ADP, a bioluminescent enzyme, and a luciferin substrate.
  • the first detection reagent comprises ADP forming succinyl-CoA ligase (SCS-ADP), ATP, and coenzyme A
  • the second detection reagent comprises an ATP depletion reagent
  • a third reagent comprises an ADP-to-ATP conversion/detection reagent; wherein the first reaction mixture is contacted sequentially with the ATP depletion reagent and the ADP-to-ATP conversion/detection reagent, and wherein the ATP depletion reagent comprises adenylate cyclase and pyrophosphatase and the ADP-to-ATP conversion/detection reagent comprises pyruvate kinase, phosphoenolpyruvate, a bioluminescent enzyme, and a luciferin substrate.
  • SCS-ADP succinyl-CoA ligase
  • ATP depletion reagent comprises adenylate cyclase and pyrophosphatas
  • the method includes contacting the sample with a peptide, protein, or non-protein substrate, 2-oxoglutarate, Fe(II), and ascorbate to form a succinate reaction mixture, wherein succinate is formed if the sample comprises 2OG-dependent dioxygenase; the first detection reagent may be added to the 2OG-dependent dioxygenase activity assay in order to prevent succinate inhibition of 2OG-dependent dioxygenase; contacting the succinate reaction mixture with a first detection reagent to form a first reaction mixture, wherein the succinate formed in step (a) is converted to succinyl-CoA or ATP; contacting the first reaction mixture with a second detection reagent to form a second reaction mixture; and detecting luminescence in the second reaction mixture, thereby detecting or determining the presence or
  • the first detection reagent comprises 3-oxoacid CoA-transferase (SCOT), inorganic phosphate, and acetoacetyl-CoA and the second detection reagent comprises succinyl-CoA ligase (SCS), e.g., SCS-ADP, ADP, a bioluminescent enzyme, and a luciferin substrate.
  • SCS succinyl-CoA ligase
  • the first detection reagent comprises 3-oxoacid CoA-transferase (SCOT), inorganic phosphate, acetoacetyl-CoA, succinyl-CoA ligase (SCS) (e.g., SCS-ADP), ADP, magnesium chloride, and the second detection reagent comprises a bioluminescent enzyme and a luciferin substrate.
  • the first detection reagent comprises SCS-GDP, GTP, and coenzyme A
  • the second detection reagent comprises guanylate kinase (GMPK), ADP, a bioluminescent enzyme, and a luciferin substrate.
  • the first detection reagent comprises SCS-GDP, GTP, coenzyme A, guanylate kinase (GMPK), and ADP
  • the second detection reagent comprises a bioluminescent enzyme, and a luciferin substrate.
  • the first detection reagent comprises ADP forming succinyl-CoA ligase (SCS-ADP), ATP, and coenzyme A
  • the second detection reagent comprises an ATP depletion reagent
  • a third reagent comprises an ADP-to-ATP conversion/detection reagent; wherein the first reaction mixture is contacted sequentially with the ATP depletion reagent and the ADP-to-ATP conversion/detection reagent, and wherein the ATP depletion reagent comprises adenylate cyclase and pyrophosphatase, and the ADP-to-ATP conversion/detection reagent comprises pyruvate kinase, phosphoenolpyruvate, a bioluminescent enzyme, and a luciferin substrate.
  • SCS-ADP succinyl-CoA ligase
  • ATP depletion reagent comprises adenylate cyclase and pyrophosphata
  • Another aspect of the present disclosure provides for methods of detecting and quantifying succinate in cellular and tissue extracts.
  • the methods involve removing any ATP present in the sample and converting the succinate present in the extract to succinyl-CoA, using any of the methods described above.
  • SCOT may be used to convert the succinate to succinyl-CoA.
  • the succinyl-CoA that is generated by the SCOT reaction may be converted by SCS-ADP to ATP, which is detected by luminescence.
  • the method includes contacting the sample with a DNA oligonucleotide containing a 5mC, a 5hmC, a 5fC or a 5caC modification, 2-oxoglutarate, Fe(II), and ascorbate to form a Tet succinate reaction mixture, wherein succinate is formed if the sample comprises Tet; contacting the Tet succinate reaction mixture with a first detection reagent to form a first reaction mixture, wherein the succinate formed in step (a) is converted to succinyl-CoA or ATP; contacting the first reaction mixture with a second detection reagent to form a second reaction mixture; and detecting luminescence in the second reaction mixture, thereby detecting or determining the presence or amount of Tet or Tet activity in the sample.
  • the first detection reagent may be added to the Tet succinate reaction mixture to prevent succinate inhibition
  • the first detection reagent comprises 3-oxoacid CoA-transferase (SCOT), inorganic phosphate, and acetoacetyl-CoA and the second detection reagent comprises succinyl-CoA ligase (SCS), e.g., SCS-ADP, ADP, a bioluminescent enzyme, and a luciferin substrate.
  • SCS succinyl-CoA ligase
  • the first detection reagent comprises 3-oxoacid CoA-transferase (SCOT), inorganic phosphate, acetoacetyl-CoA, succinyl-CoA ligase (SCS) (e.g., SCS-ADP), ADP, magnesium chloride, and the second detection reagent comprises a bioluminescent enzyme and a luciferin substrate.
  • the first detection reagent comprises SCS-GDP, GTP, and coenzyme A
  • the second detection reagent comprises guanylate kinase (GMPK), ADP, a bioluminescent enzyme, and a luciferin substrate.
  • the first detection reagent comprises ADP forming succinyl-CoA ligase (SCS-ADP), ATP, and coenzyme A
  • the second detection reagent comprises an ATP depletion reagent
  • a third reagent comprises an ADP-to-ATP conversion/detection reagent; wherein the first reaction mixture is contacted sequentially with the ATP depletion reagent and the ADP- to-ATP conversion/detection reagent, and wherein the ATP depletion reagent comprises adenylate cyclase and pyrophosphatase, and the ADP-to-ATP conversion/detection reagent comprises pyruvate kinase, phosphoenolpyruvate, a bioluminescent enzyme, and a luciferin substrate.
  • SCS-ADP succinyl-CoA ligase
  • ATP depletion reagent comprises adenylate cyclase and pyrophosphata
  • the herein described methods for detecting or determining the presence or amount of succinate may be incorporated into methods of screening candidate modulators of 2-oxoglutarate oxygenase activity.
  • the herein described methods of screening for modulators of 2-oxoglutarate oxygenase activity make use of the generation of measurable luminescence, wherein the luminescence (relative light units; RLUs) measured is proportional to the level of succinate in the sample and the 2-oxoglutarate oxygenase activity.
  • the sample may be contacted with a compound of interest.
  • the methods include contacting the sample with the compound to form a test sample, wherein the sample comprises a 2-oxoglutarate oxygenase; contacting the test sample with a peptide, protein, or non-protein substrate, 2-oxoglutarate, Fe(II), and ascorbate to form a succinate reaction mixture; contacting the succinate reaction mixture with a first detection reagent to form a first reaction mixture, whereby the succinate formed in step (a) is converted to succinyl-CoA or ATP; contacting the first reaction mixture with a second or ATP detection reagent to form a second reaction mixture; detecting luminescence in the second reaction mixture; and comparing the luminescence in the second reaction mixture to luminescence in a control sample, wherein if the luminescence in the second reaction mixture is different from the luminescence in a control sample, the compound is identified as being a modulator of 2-oxoglutarate oxygenase activity.
  • the first detection reagent comprises 3-oxoacid CoA-transferase (SCOT), inorganic phosphate, and acetoacetyl-CoA
  • the second or ATP detection reagent comprises succinyl-CoA ligase (SCS), e.g., SCS-ADP, ADP, a bioluminescent enzyme, and a luciferin substrate.
  • SCS succinyl-CoA ligase
  • the first detection reagent comprises 3-oxoacid CoA-transferase (SCOT), inorganic phosphate, acetoacetyl-CoA, succinyl-CoA ligase (SCS) (e.g., SCS-ADP), ADP, magnesium chloride, and the second detection reagent comprises a bioluminescent enzyme and a luciferin substrate.
  • the first detection reagent comprises SCS-GDP, GTP, and coenzyme A
  • the second or ATP detection reagent comprises guanylate kinase (GMPK), ADP, a bioluminescent enzyme, and a luciferin substrate.
  • the first detection reagent comprises SCS-GDP, GTP, coenzyme A, guanylate kinase (GMPK), and ADP
  • the second detection reagent comprises a bioluminescent enzyme, and a luciferin substrate.
  • the first detection reagent comprises ADP forming succinyl-CoA ligase (SCS-ADP), ATP, and coenzyme A
  • the second detection reagent comprises an ATP depletion reagent
  • a third reagent comprises an ADP-to-ATP conversion/detection reagent; wherein the first reaction mixture is contacted sequentially with the ATP depletion reagent and the ADP-to-ATP conversion/detection reagent, and wherein the ATP depletion reagent comprises adenylate cyclase and pyrophosphatase, and the ADP-to-ATP conversion/detection reagent comprises pyruvate kinase, phosphoenolpyruvate, a bioluminescent enzyme, and a luciferin substrate.
  • SCS-ADP succinyl-CoA ligase
  • ATP depletion reagent comprises adenylate cyclase and pyrophosphata
  • the methods include contacting the sample with the compound to form a test sample, wherein the sample comprises a JMJC demethylase; contacting the test sample with a peptide or protein substrate, 2-oxoglutarate, Fe(II), and ascorbate to form a succinate reaction mixture; contacting the succinate reaction mixture with a first detection reagent to form a first reaction mixture, whereby the succinate formed in step (a) is converted to succinyl-CoA or ATP; contacting the first reaction mixture with an second or ATP detection reagent to form a second reaction mixture; detecting luminescence in the second reaction mixture; and comparing the luminescence in the second reaction mixture to luminescence in a control sample, wherein if the luminescence in the second reaction mixture is different from the luminescence in a control sample, the compound is identified as being a modulator of JMJC demethyla
  • the first detection reagent comprises 3-oxoacid CoA-transferase (SCOT), inorganic phosphate, and acetoacetyl-CoA and the second or ATP detection reagent comprises succinyl-CoA ligase (SCS), e.g., SCS-ADP, ADP, a bioluminescent enzyme, and a luciferin substrate.
  • SCS succinyl-CoA ligase
  • the first detection reagent comprises 3-oxoacid CoA-transferase (SCOT), inorganic phosphate, acetoacetyl-CoA, succinyl-CoA ligase (SCS) (e.g., SCS-ADP), ADP, magnesium chloride, and the second detection reagent comprises a bioluminescent enzyme and a luciferin substrate.
  • the first detection reagent comprises SCS-GDP, GTP, and coenzyme A
  • the second or ATP detection reagent comprises guanylate kinase (GMPK), ADP, a bioluminescent enzyme, and a luciferin substrate.
  • the first detection reagent comprises SCS-GDP, GTP, coenzyme A, guanylate kinase (GMPK), and ADP
  • the second detection reagent comprises a bioluminescent enzyme, and a luciferin substrate.
  • the first detection reagent comprises ADP forming succinyl-CoA ligase (SCS-ADP), ATP, and coenzyme A
  • the second detection reagent comprises an ATP depletion reagent
  • a third reagent comprising an ADP-to-ATP conversion/detection reagent; wherein the first reaction mixture is contacted sequentially with the ATP depletion reagent, and the ADP- to-ATP conversion/detection reagent, and wherein the ATP depletion reagent comprises adenylate cyclase and pyrophosphatase, and the ADP-to-ATP conversion/detection reagent comprises pyruvate kinase, phosphoenolpyruvate, a bioluminescent enzyme, and a luciferin substrate.
  • SCS-ADP succinyl-CoA ligase
  • ATP depletion reagent comprises adenylate cyclase and pyro
  • the methods include contacting the sample with the compound to form a test sample, wherein the sample comprises a 2OG-dependent dioxygenase; contacting the test sample with a peptide, protein, or non-protein substrate, 2-oxoglutarate, Fe(II), and ascorbate to form a succinate reaction mixture; contacting the succinate reaction mixture with a first detection reagent to form a first reaction mixture, whereby the succinate formed in step (a) is converted to succinyl-CoA or ATP; contacting the first reaction mixture with a second or ATP detection reagent to form a second reaction mixture; detecting luminescence in the second reaction mixture; and comparing the luminescence in the second reaction mixture to luminescence in a control sample, wherein if the luminescence in the second reaction mixture is different from the luminescence in a control sample, the compound is identified as being
  • the first detection reagent comprises 3-oxoacid CoA-transferase (SCOT), inorganic phosphate, and acetoacetyl-CoA and the second or ATP detection reagent comprises succinyl-CoA ligase (SCS), e.g., SCS-ADP, ADP, a bioluminescent enzyme, and a luciferin substrate.
  • SCS succinyl-CoA ligase
  • the first detection reagent comprises 3-oxoacid CoA-transferase (SCOT), inorganic phosphate, acetoacetyl-CoA, succinyl-CoA ligase (SCS) (e.g., SCS-ADP), ADP, magnesium chloride, and the second detection reagent comprises a bioluminescent enzyme and a luciferin substrate.
  • the first detection reagent comprises SCS-GDP, GTP, and coenzyme A
  • the second or ATP detection reagent comprises guanylate kinase (GMPK), ADP, a bioluminescent enzyme, and a luciferin substrate.
  • the first detection reagent comprises SCS-GDP, GTP, coenzyme A, guanylate kinase (GMPK), and ADP
  • the second detection reagent comprises a bioluminescent enzyme, and a luciferin substrate.
  • the first detection reagent comprises ADP forming succinyl-CoA ligase (SCS-ADP), ATP, and coenzyme A
  • the second detection reagent comprises an ATP depletion reagent
  • a third reagent comprising an ADP-to-ATP conversion/detection reagent
  • the first reaction mixture is contacted sequentially with the ATP depletion reagent and the ADP- to-ATP conversion/detection reagent
  • the ATP depletion reagent comprises adenylate cyclase and pyrophosphatase
  • the ADP-to-ATP conversion/detection reagent comprises pyruvate kinase, phosphoenolpyruvate, a bioluminescent enzyme, and a luciferin substrate.
  • a variety of different types of libraries of candidate modulator compounds can be used and screened in the method of the present disclosure.
  • a candidate modulator may be an antibody, a small molecule, a drug, a peptide, a nucleic acid, an oligosaccharide, or an inorganic compound.
  • An identified modulator compound may be derived from a library of candidate modulator compounds.
  • a library of compounds may be a combinatorial library. The method may comprise stimulating a host cell to express the candidate modulator compound.
  • the candidate modulators may be compared with selective inhibitors, such as histone demethylase JMJD inhibitors JIB-04 (E)-N-(5-Chloro-pyridin-2-yl)-N'-(phenyl-pyridin-2-yl-methylene)-hydrazine) or IOX 1 (5-carboxy-8HQ; also known as 8-Hydroxy-5-quinolinecarboxylic acid).
  • selective inhibitors such as histone demethylase JMJD inhibitors JIB-04 (E)-N-(5-Chloro-pyridin-2-yl)-N'-(phenyl-pyridin-2-yl-methylene)-hydrazine) or IOX 1 (5-carboxy-8HQ; also known as 8-Hydroxy-5-quinolinecarboxylic acid).
  • Modulators identified by the herein described method may be compounds showing pharmacological activity or therapeutic activity.
  • Compounds with pharmacological activity are able to enhance or interfere with the activity of a JMJC demethylase or a fragment thereof.
  • the compounds having the desired pharmacological activity may be administered in a physiologically acceptable carrier to a host.
  • the agents may be administered in a variety of ways, orally, parenterally e.g., subcutaneously, intraperitoneally, intravascularly, etc. Depending upon the manner of introduction, the compounds may be formulated in a variety of ways.
  • the concentration of a therapeutically active compound in the formulation may vary from about 0.1-100 wt%.
  • Modulators of the present disclosure can be administered at a rate determined by the LD50 of the modulator, and the side-effects of the modulator at various concentrations, as applied to the mass and overall health of the subject. Administration can be accomplished via single or divided doses.
  • the identified modulators of the disclosure may be used alone or in conjunction with other agents that are known to be beneficial in treating or preventing human diseases that are mediated by DNA methylation, demethylation, and/or 5-mC hydroxylation.
  • the modulators of the disclosure and another agent may be co-administered, either in concomitant therapy or in a fixed combination, or they may be administered at separate times.
  • control sample may be a sample that has not been contacted with the compound.
  • the control sample may be analyzed concurrently with the test sample, as described above.
  • the results obtained from the test sample may be compared to the results obtained from the control sample.
  • Standard curves may be provided, with which assay results for the test sample may be compared. Such standard curves present levels of marker as a function of assay units, i.e. luminescent signal intensity.
  • the luminescence generated by a luciferase-luciferin reaction is typically detected with a luminometer although other detection means may be used.
  • the presence of light greater than background level indicates the presence of ATP, and thus succinate and/or 2-oxoglutarate oxygenase, in the sample.
  • the background level of luminescence is typically measured in the same matrix, but in the absence of the sample. Suitable control reactions are readily designed by one of skill in the art.
  • Luciferases may allow for multiple analyses of a sample over time or analysis of many samples over time.
  • the luciferases used in the compositions and methods of the invention have enhanced thermostability and/or chemostability properties.
  • a full length luciferase, 2-oxoglutarate oxygenase, such as JMJC demethylase or 2OG-dependent dioxygenase, 3-oxoacid CoA-transferase, or succinate-CoA ligase variant will have at least about 80% amino acid sequence identity, at least about 81% amino acid sequence identity, such as at least about 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity with a corresponding full-length native luciferase, 2-oxoglutarate oxygenase, such as JMJC demethylase or 2OG-depedent dioxygenase, SCOT, or SCS.
  • variant fragments are at least about 50 amino acids in length, often at least about 60 amino acids in length, more often at least about 70, 80, 90, 100, 150, 200, 300, 400, 500 or 550 amino acids in length, or more and retain the ability to generate luminescence, transfer phosphate groups, transfer glucose, and hydroxylate.
  • a full length luciferase, 2-oxoglutarate oxygenase, such as JMJC demethylase or 2OG-dependent dioxygenase, SCOT, SCS, fragment thereof, or variant thereof may be fused to heterologous amino acid sequences and still be functional in the invention.
  • full length beetle luciferase, 2-oxoglutarate oxygenase, such as JMJC demethylase or 2OG-dependent dioxygenase, SCOT, or SCS, fragments thereof or variants thereof used in the compositions and methods of the present invention may be purified from a native source or prepared by a number of techniques, including (1) chemical synthesis, (2) enzymatic (protease) digestion of luciferase, and (3) recombinant DNA methods. Chemical synthesis methods are well known in the art, as are methods that employ proteases to cleave specific sites.
  • DNA encoding the enzymes, variants and fragments may be prepared and then expressed in a host organism, such as E. coli. Methods such as endonuclease digestion or polymerase chain reaction (PCR) allow one of skill in the art to generate an unlimited supply of well-defined fragments.
  • PCR polymerase chain reaction
  • Any type of amino acid substitution, insertion or deletion, or combination thereof may be used to generate a variant luciferase, 2-oxoglutarate oxygenase, such as JMJC demethylase or 2OG-dependent dioxygenase, SCOT, or SCS with a conservative amino acid substitution is more likely to retain activity.
  • Conservative substitutions whereby an amino acid of one class is replaced with another amino acid of the same type fall within the scope of the disclosure if the substitution does not impair enzyme activity.
  • Variant luciferase, 2-oxoglutarate oxygenase, such as JMJC demethylase or 2OG-dependent dioxygenase, SCOT, or SCS genes or gene fragments may be made using methods known in the art such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis (site-directed mutagenesis) cassette mutagenesis, restriction selection mutagenesis, PCR mutagenesis or other known techniques can be performed on the cloned DNA to produce the variant DNA.
  • the disclosed methods may be used with any sample that is suspected of containing succinate or derivatives thereof.
  • the disclosed methods may be used with non-biological or biological samples.
  • Succinate and derivatives thereof are used for the manufacturing of various industrial products, such as products made in the pharmaceutical or cosmetic industries.
  • the disclosed methods may be used in testing in the manufacturing of pharmaceuticals, cosmetics, detergents, surfactants, plastics, polymers, lubricants, and resins (paint).
  • the disclosed methods may be used in food and beverage, such as wine, testing where succinate is used as a quality indicator.
  • the disclosed methods may be used with samples containing biological components.
  • the sample may comprise cells and/or tissue.
  • the sample may comprise heterogeneous mixtures of components (including intact cells, cell extracts, cell lysates, bacteria, viruses, organelles, and mixtures thereof) or a single component or homogeneous group of components (e.g., natural or synthetic amino acid, nucleic acid or carbohydrate polymers, or lipid membrane complexes).
  • the compounds are generally non-toxic to living cells and other biological components within the concentrations of use.
  • the sample may include an animal (e.g., a vertebrate), a plant, a fungus, physiological fluid (e.g., blood, plasma, urine, mucous secretions and the like), a cell, a cell lysate, a cell supernatant, or a purified fraction of a cell (e.g., a subcellular fraction).
  • the sample may be a cell.
  • the sample may be a live cell.
  • the cell may be a eukaryotic cell, e.g., yeast, avian, plant, insect or mammalian cells, including but not limited to human, simian, murine, canine, bovine, equine, feline, ovine, caprine or swine cells, or prokaryotic cells, or cells from two or more different organisms, or cell lysates or supernatants thereof.
  • yeast eukaryotic cell
  • avian e.g., plant, insect or mammalian cells
  • insect or mammalian cells including but not limited to human, simian, murine, canine, bovine, equine, feline, ovine, caprine or swine cells, or prokaryotic cells, or cells from two or more different organisms, or cell lysates or supernatants thereof.
  • the cells may not have been genetically modified via recombinant techniques (non-recombinant cells), or may be recombinant cells which are transiently transfected with recombinant DNA and/or the genome of which is stably augmented with a recombinant DNA, or which genome has been modified to disrupt a gene, e.g., disrupt a promoter, intron or open reading frame, or replace one DNA fragment with another.
  • the recombinant DNA or replacement DNA fragment may encode a molecule to be detected by the methods of the invention, a moiety which alters the level or activity of the molecule to be detected, and/or a gene product unrelated to the molecule or moiety that alters the level or activity of the molecule.
  • the cells may have been genetically modified via recombinant techniques.
  • the sample may include a succinate forming or generating enzyme.
  • the succinate forming or generating enzyme may be 2-oxoglutarate oxygenase, such as a JMJC demethylase or 2OG-dependent dioxygenase, as described above.
  • the succinate forming or generating enzyme may be a native or recombinant a succinate forming or generating enzyme.
  • the sample may include a native or recombinant demethylase.
  • the succinate forming or generating enzyme is purified or isolated.
  • the sample may include a purified or isolated demethylase.
  • Methods and compositions may involve a purified, or substantially pure, substrates, such as a peptide, protein, or non-protein substrates, 2-oxoglutarate, Fe(II), ascorbate, inorganic phosphate, acetoacetyl-CoA, ADP, and a luciferin substrate, and/or enzyme, such as bioluminescent enzyme, such as a luciferase, 2-oxoglutarate oxygenase, such as JMJC demethylase or 2OG-dependent dioxygenase, SCOT, and SCS.
  • substrates such as a peptide, protein, or non-protein substrates, 2-oxoglutarate, Fe(II), ascorbate, inorganic phosphate, acetoacetyl-CoA, ADP, and a luciferin substrate
  • enzyme such as bioluminescent enzyme, such as a luciferase, 2-oxoglutarate oxygenase, such as
  • purification may result in a molecule that is about or at least about 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7 99.8, 99.9% or more pure, or any range derivable therein, relative to any contaminating components (w/w or w/v).
  • Kits for analysis of succinate are provided herein.
  • Such kits comprise reagents for analysis of succinate.
  • Such kits may comprise an active SCOT and active SCS-ADP; active SCS-GDP and active guanylate kinase; and/or active SCS-ADP, active adenylate cyclase, active pyrophosphatase, and active pyruvate kinase.
  • Such kits may further comprise one or more reference succinate samples; a buffer, instructions, a bioluminescent enzyme and a luciferin substrate.
  • kit components, compositions and buffers may also be modified by the addition of suitable components, such as ADP, GDP, ATP, inorganic phosphate, acetoacetyl-CoA, a divalent cation, such as magnesium, for example magnesium chloride, calmodulin, phosphoenolpyruvate, salts, chelators, etc.
  • suitable kit components, compositions and buffers that may be used in the described methods may also be obtained commercially.
  • the different components may comprise subsets of these parts and may be combined in any way that either facilitates the application of the invention or prolongs storage life.
  • Kits for analysis of a succinate forming or generating enzyme are provided herein.
  • Such kits comprise reagents for analysis of the succinate forming or generating enzyme.
  • Such kits comprise reagents for analysis of succinate, as described above, a peptide, protein, or non-protein substrate, 2-oxoglutarate, Fe(II), one or more reference succinate samples; a buffer, instructions, a bioluminescent enzyme and a luciferin substrate.
  • the kit components, compositions and buffers may also be modified by the addition of suitable components, such as ADP, inorganic phosphate, acetoacetyl-CoA salts, chelators, etc.
  • kits components, compositions and buffers that may be used in the described methods such as peptide, protein, or non-protein substrates, 2-oxoglutarate and Fe(II), may also be obtained commercially.
  • the different components may comprise subsets of these parts and may be combined in any way that either facilitates the application of the invention or prolongs storage life.
  • the kit comprises a separate container comprising lyophilized luciferase.
  • the container comprising lyophilized luciferase further comprises lyophilized luciferin or a derivative thereof that is a luciferase substrate.
  • One or more reagents may be supplied in a solid form or liquid buffer that is suitable for inventory storage, and later for addition into the reaction medium when the method of using the reagent is performed. Suitable packaging is provided.
  • the reagents included in the kits may be supplied in containers of any sort such that the life of the different components are preserved and are not adsorbed or altered by the materials of the container.
  • sealed glass ampules may contain lyophilized luciferase or buffer that has been packaged under a neutral, non-reacting gas, such as nitrogen.
  • Ampules may consist of any suitable material, such as glass, organic polymers, such as polycarbonate, polystyrene, etc., ceramic, metal or any other material typically employed to hold reagents.
  • suitable containers include simple bottles that may be fabricated from similar substances as ampules, and envelopes, that may consist of foil-lined interiors, such as aluminum or an alloy.
  • Containers include test tubes, vials, flasks, bottles, syringes, or the like.
  • Containers may have a sterile access port such as a bottle having a stopper that may be pierced by a hypodermic injection needle.
  • Other containers may have two compartments that are separated by a readily removable membrane that upon removal permits the components to mix.
  • Removable membranes may be glass, plastic, rubber, etc.
  • kits may also be supplied with instructional materials. Instructions may be printed on paper or other substrate and/or may be supplied as an electronic-readable medium such as a floppy disc, CD-ROM, DVD-ROM, Zip disc, videotape, audio tape, etc. Detailed instructions may not be physically associated with the kit; instead, a user may be directed to an internet web site specified by the manufacturer or distributor of the kit, or supplied as electronic mail.
  • the present invention may be utilized as illustrated by the following non-limiting examples.
  • Bioluminescent Succinate Detection Reagent I (SCS-GDP/GMPK method) 3.2 ⁇ L of Bioluminescent Succinate Detection Solution I (SCS-GDP) in 1 mL of 50 mM Tris-HCl, pH 7.5, and 10 mM MgCl 2 Bioluminescent Succinate Detection Solution II (SCS-GDP/GMPK method) 100 ⁇ g/mL of guanylate kinase in water Bioluminescent Succinate Detection Reagent II (SCS-GDP/GMPK method) 1.6 ⁇ L of Bioluminescent Succinate Detection Solution II (GMPK) in 1 mL of Kinase-Glo® Reagent Method 3 Bioluminescent Succinate Detection Solution I (SCS-ADP/ADP-GloTM method) 220 U/mL of SCS-ADP in ammonium sulfate Bioluminescent Succinate Detecti
  • the 3-Oxoacid CoA-Transferase gene of Sus scrofa was cloned as a synthetic codon-optimized sequence from Genscript.
  • the expressed protein ranged from amino acid 40 to 517 and included an 8X His tag on the C-terminus.
  • the protein was expressed in E. coli KRX cells (Promega) from the T7 promoter of Flexi vector pF1K (Promega).
  • the SCOT enzyme was purified over HisLink resin (Promega), and the purified eluate was dialyzed into 25 mM HEPES, pH 7.5, 25 mM NaCl, 1 mM DTT, and 50% glycerol. The size of the purified enzyme was verified via SDS-PAGE.
  • SCS-ADP and SCS-GDP The Succinyl-CoA Synthetase alpha and beta subunit genes of Sus scrofa (SCS-ADP and SCS-GDP) were cloned as synthetic codon-optimized sequences from Genscript.
  • the SCS alpha protein includes a 6X His tag on the N-terminus.
  • the proteins were expressed in E . coli KRX cells (Promega) from the T7 promoter of Flexi vector pF1K (Promega).
  • the SCS-ADP and SCS-GDP enzymes were purified over HisLink resin (Promega), and the purified eluates were dialyzed into 25 mM HEPES, pH 7.5, 25 mM NaCl, 1 mM DTT. The size of the purified enzymes was verified via SDS-PAGE.
  • E. coli (SCS-ADP) was cloned directly out of W3110 E. coli genomic DNA a single bi-cistronic sequence.
  • the alpha protein includes a 6X His tag on the C-terminus. Expression and purification were performed as above.
  • the succinate detection assay in FIG. 2 was performed generally in two steps as follows.
  • the succinate titration was performed in 25 ⁇ L containing 1x Demethylase Reaction Buffer (50 mM HEPES, pH 7.5, 0.01% Tween-20, 10 ⁇ g/mL BSA, 100 ⁇ M ascorbate, 10 ⁇ M Fe(II), 10 ⁇ M ⁇ -ketoglutarate).
  • 25 ⁇ L of Bioluminescent Succinate Detection Reagent I was added to the succinate samples, and the mixture was incubated for 60 min at room temperature ( ⁇ 23°C).
  • the method of the present invention was used to assay representative demethylating enzymes including, but not limited to, JMJD2C (BPS Bioscience, cat # 50105) with substrate Lys(Me3)9-histone H3 (1-21) (Anaspec, cat # 64452), JARID1A (BPS Bioscience, cat # 50110) with substrate Lys(Me3)4-histone H3 (1-21) (Anaspec, cat # 64194) and UTX (Cayman Chemical, cat # 10774) with substrate Lys(Me3)27-histone H3 (23-34) (Anaspec, cat # 64378).
  • Each enzyme was titrated separately, and the reaction was performed in the Demethylase Reaction Buffer.
  • the method of the present invention may be used to screen for compounds that alter the activity of all enzymes involved in histone demethylation.
  • reactions with JMJC demethylase enzymes including but not limited to JMJD2C (BPS Bioscience, cat # 50105), were carried out at concentrations up to 50 ⁇ M of histone H3 peptide substrates Lys(Me3)9-histone H3 (1-21) (Anaspec, cat # 64452), Lys(Me2)9-histone H3 (1-21) (Anaspec, cat # 65401), and histone H3 (1-21) (Anaspec, cat # 61701).
  • the amount of luminescence generated was proportional to substrate concentration until saturation was reached and was proportional to the amount of enzyme within the linear range of the reaction.
  • reactions with JMJC demethylase enzymes including but not limited to JMJD2C (BPS Bioscience, cat # 50105), were carried out at concentrations up to 10 ⁇ M of Lys(Me3)9-histone H3 (1-21) peptide substrate (Anaspec, cat # 64452).
  • the amount of luminescence generated was proportional to substrate concentration until saturation was reached and was proportional to the amount of enzyme within the linear range of the reaction.
  • Succinate in the samples was converted to succinyl-CoA using a Bioluminescent Succinate Detection Reagent I.
  • the succinyl-CoA generated was then converted to ATP, and the ATP was detected using the Bioluminescent Succinate Detection Reagent II. Briefly, 5 ⁇ L of JMJC demethylase in 50 mM HEPES, pH 7.5, 0.01% Tween-20, and 10 ⁇ g/mL BSA was added to wells of a 96-well low volume plate. Titrations of JMJC demethylase inhibitors (amounts indicated on figures) were performed in a reaction buffer containing 50 mM HEPES, pH 7.5, 0.01% Tween-20, 10 ⁇ g/mL BSA, and 5% DMSO (v/v) and incubated for 10 min at room temperature.
  • the reaction was initiated with the addition of 4 ⁇ L of 25 ⁇ M histone H3 Me3K9 peptide substrate diluted in a reaction buffer containing 50 mM HEPES, pH 7.5, 0.01% Tween-20, 10 ⁇ g/mL BSA, 250 ⁇ M ascorbate, 1.5 ⁇ M Fe(II), and 62.5 ⁇ M ⁇ -ketoglutarate. JMJC demethylase reactions were carried out in 10 ⁇ L for 60 min at room temperature.
  • the succinate detection assay described in FIG. 8 was performed generally in two steps as follows.
  • the succinate titration was performed in 25 ⁇ L containing 50 mM Tris-HCl, pH 7.5, 10 mM MgCl 2 , 15 ⁇ M coenzyme A, and 15 ⁇ M GTP.
  • 25 ⁇ L of Bioluminescent Succinate Detection Reagent I (SCS-GDP/GMPK method) was added to the succinate samples, and the mixture was incubated for 60 min at room temperature.
  • the method of the present invention was used to assay representative demethylating enzymes including, but not limited to, JMJD2C (BPS Bioscience, cat # 50105) and JMJD2A (BPS Bioscience, cat # 50123), and peptide Lys(Me3)9-histone H3 (1-21) (Anaspec, cat # 64452) used as substrate.
  • Each enzyme was titrated separately, and the reaction performed in Demethylase Reaction Buffer without Tween-20 and BSA (50 mM HEPES, pH 7.5, 100 ⁇ M ascorbate, 5 ⁇ M Fe(II), and 5 ⁇ M ⁇ -ketoglutarate).
  • Succinate and GTP were converted to succinyl-CoA and GDP using Bioluminescent Succinate Detection Reagent I (SCS-GDP/GMPK method) followed by the conversion of GDP and ADP to ATP and GMP. ATP was detected using the Bioluminescent Succinate Detection Reagent II (SCS-GDP/GMPK method). Briefly, 6 ⁇ L of serially diluted JMJC demethylase in 50 mM HEPES, pH 7.5 was added to wells of a 96-well low volume plate.
  • the JMJC demethylase reaction was started by adding 6 ⁇ L of a mixture containing Demethylase Reaction Buffer without Tween-20 and BSA, and 2.5 ⁇ M trimethylated histone H3 peptide substrate. After incubation of 60 min at room temperature, 12 ⁇ L of the succinate to succinyl-CoA conversion reagent (Bioluminescent Succinate Detection Reagent I (SCS-GDP/GMPK method)) was added, mixed for 2 min on an orbital shaker, and then incubated at room temperature for 60 min.
  • SCS-GDP/GMPK method Bioluminescent Succinate Detection Reagent I
  • reactions with JMJC demethylase enzymes including, but not limited to JMJD2C (BPS Bioscience, cat # 50105), were carried out at concentrations up to 50 ⁇ M of histone H3 peptide substrates Lys(Me3)9-histone H3 (1-21) (Anaspec, cat # 64452), Lys(Me2)9-histone H3 (1-21) (Anaspec, cat # 65401), and histone H3 (1-21) (Anaspec, cat # 61701), and the amount of luminescence generated was proportional to substrate concentration until saturation was reached and was proportional to the amount of enzyme within the linear range of the reaction.
  • the succinate detection assay described in FIG. 12 was performed generally in three steps as follows herein.
  • the succinate titration was performed in 10 ⁇ L containing 50 mM Tris-HCl, pH 7.5.
  • 10 ⁇ L of Bioluminescent Succinate Detection Reagent I (SCS-ADP/ADP-GloTM method, containing 50 mM Tris-HCl, pH 7.5, 10 mM MgCl 2 , 30 ⁇ M ATP, and 30 ⁇ M coenzyme A) was added to the succinate samples, and the mixture was incubated for 60 min at room temperature.
  • ADP-GloTM Reagent 20 ⁇ L was added to the succinate samples, and the mixture was incubated for 40 min at room temperature.
  • ADP-GloTM Kinase Detection Reagent 40 ⁇ L was added, and the mixture was incubated for 30 min at room temperature before luminescence was detected on a luminometer.
  • the succinate titration described in FIG. 13 was performed in wells of 96-well low volume plates.
  • the method of the present invention was used to assay representative demethylating enzymes including, but not limited to JMJD2C (BPS Bioscience, cat # 50105) and JMJD2A (BPS Bioscience, cat # 50123), and peptide Lys(Me3)9-histone H3 (1-21) (Anaspec, cat # 64452) used as substrate.
  • Each enzyme was titrated separately and together, and the reaction performed in Demethylase Reaction Buffer without Tween-20 and BSA (50 mM HEPES, pH 7.5, 100 ⁇ M ascorbate, 5 ⁇ M Fe(II), and 5 ⁇ M ⁇ -ketoglutarate).
  • Succinate and ATP were converted to succinyl-CoA and ADP using Bioluminescent Succinate Detection Reagent I (SCS-ADP/ADP-GloTM method).
  • ADP-GloTM Reagent was added to the wells of a 96-well low volume plate to remove any remaining ATP from the reaction, followed by the conversion of ADP to ATP and detection of ATP using the ADP-GloTM Kinase Detection Reagent. Briefly, 6 ⁇ L of serially diluted JMJC demethylase in 50 mM HEPES, pH 7.5 was added to wells of a 96-well low volume plate.
  • the JMJC demethylase reaction was started by adding 6 ⁇ L of a mixture containing Demethylase Reaction Buffer without Tween-20 and BSA and 2.5 ⁇ M trimethylated histone H3 peptide substrate. After incubation of 60 min at room temperature, 12 ⁇ L of the succinate to succinyl-CoA conversion reagent (Bioluminescent Succinate Detection Reagent I (SCS-ADP/ADP-GloTM method)) was added, mixed for 2 min on an orbital shaker, and then incubated at room temperature for 60 min. To the samples, 24 ⁇ L of ADP-GloTM Reagent was added, and the samples incubated at room temperature for 40 min.
  • the method of the present invention may be used to screen for compounds that alter the activity of the all enzymes involved in histone demethylation.
  • the method of the present invention was used to determine the presence and/or amount of succinate in tissue extracts. Any ATP present in the sample was removed. After removal of any ATP present in the sample, the succinate was converted to succinyl-CoA by SCOT, the ADP was converted to ATP, and the ATP was detected using the Bioluminescent Succinate Detection Reagent II (ADP-to-ATP Conversion Reagent). The ATP depleted samples that were tested for conversion of succinate (containing SCOT and SCS) were compared to samples which contained only Luciferase/luciferin to assess the succinate presence.
  • ATP-depleted cell extract in 50 mM Potassium Phosphate, pH 7.5, 10mM MgCl 2 was incubated with 10 ⁇ L of the succinate to succinyl-CoA conversion reagent (Bioluminescent Succinate Detection Reagent I), mixed for 2 min on an orbital shaker, and then incubated at room temperature for 60 min.
  • Bioluminescent Succinate Detection Reagent II was added, and the samples were incubated at room temperature for 60 min. Luminescence was then detected on a luminometer ( FIG. 15 ).
  • the succinate detection assay described in FIG. 16 was performed generally in two steps as follows.
  • the succinate titration was performed in 25 ⁇ L containing 1x Demethylase Reaction Buffer (50 mM HEPES, pH 7.5, 100 ⁇ M ascorbate, 10 ⁇ M Fe(II), 10 ⁇ M ⁇ -ketoglutarate).
  • 25 ⁇ L of Bioluminescent Succinate Detection Reagent I was added to the succinate samples, and the mixture was incubated for 60 min at room temperature ( ⁇ 23°C).
  • To detect ATP 50 ⁇ L of Bioluminescent Succinate Detection Reagent II was added, and the mixture was incubated for 10 min at room temperature before luminescence was detected on a luminometer.
  • the succinate titration shown in FIG. 17 was performed in 96-well low volume plates (Corning Costar®, cat# 3693).
  • the method of the present invention was used to assay representative demethylating enzymes including, but not limited to, JMJD2C (BPS Bioscience, cat # 50105) with substrate Lys(Me3)9-histone H3 (1-21) (Anaspec, cat # 64452) and JARID1A (BPS Bioscience, cat # 50110) with substrate Lys(Me3)4-histone H3 (1-21) (Anaspec, cat # 64194).
  • Each enzyme was titrated separately, and the reaction was performed in the Demethylase Reaction Buffer.
  • Succinate was converted to ATP using Bioluminescent Succinate Detection Reagent I followed by the detection of ATP using the Bioluminescent Succinate Detection Reagent II.
  • JMJC demethylase reaction was started by adding 5 ⁇ L of a mixture containing 2x Demethylase Reaction Buffer and 20 ⁇ M trimethylated histone H3 peptide substrate. After incubation of 60 min at room temperature, 10 ⁇ L of the succinate to ATP conversion reagent (Bioluminescent Succinate Detection Reagent I) was added, mixed for 2 min on an orbital shaker, and then incubated at room temperature for 60 min.
  • the method of the present invention may be used to screen for compounds that alter the activity of all enzymes involved in histone demethylation.
  • the method of the present invention was used to assay representative hydroxylating enzymes including, but not limited to, ELGN1 (OriGene Technologies, cat # TP315158) and EGLN2 (OriGene Technologies, cat # TP306152) with substrate HIF-1 ⁇ (556-574) (Anaspec, cat # AS-61528). Each enzyme was titrated separately, and the reaction was performed in 1X Reaction Buffer (50 mM HEPES, pH 7.5, 100 ⁇ M ascorbate, 10 ⁇ M Fe(II), 10 ⁇ M ⁇ -ketoglutarate).
  • 1X Reaction Buffer 50 mM HEPES, pH 7.5, 100 ⁇ M ascorbate, 10 ⁇ M Fe(II), 10 ⁇ M ⁇ -ketoglutarate.
  • the method of the present invention may be used to screen for compounds that alter the activity of all enzymes involved in HIF1-alpha hydroxylation.

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Family Cites Families (39)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE2834704A1 (de) 1978-08-08 1980-02-21 Boehringer Mannheim Gmbh Verfahren zur quantitativen enzymatischen bestimmung von adp
US5316907A (en) 1993-01-22 1994-05-31 Regents Of The University Of Minnesota Enzymatic fluorometric assay for adenylate cyclase
WO1994017198A1 (en) 1993-01-22 1994-08-04 Regents Of The University Of Minnesota Enzymatic fluorometric assay for adenylate cyclase
GB9501170D0 (en) 1994-03-23 1995-03-08 Secr Defence Luciferases
CN1091804C (zh) 1994-07-13 2002-10-02 大不列颠及北爱尔兰联合王国国防大臣 微生物检测法和试剂
GB9417593D0 (en) 1994-09-01 1994-10-19 Secr Defence Luciferase labelling method
EP0804587B1 (en) 1995-01-20 2004-08-18 The Secretary of State for Defence Mutant luciferases
JP3409962B2 (ja) * 1996-03-04 2003-05-26 キッコーマン株式会社 生物発光試薬及びその試薬を用いたアデノシンリン酸エステルの定量法並びにその試薬を用いたａｔｐ変換反応系に関与する物質の定量法
GB9707486D0 (en) 1997-04-11 1997-05-28 Secr Defence Enzyme assays
US6602677B1 (en) 1997-09-19 2003-08-05 Promega Corporation Thermostable luciferases and methods of production
JP4537573B2 (ja) 1997-09-19 2010-09-01 プロメガ・コーポレイシヨン 熱安定ルシフェラーゼと生産方法
GB9823468D0 (en) 1998-10-28 1998-12-23 Secr Defence Novel enzyme
GB9902659D0 (en) 1999-02-05 1999-03-31 Microbiological Res Authority Assay with reduced background
JP3059435B1 (ja) 1999-03-18 2000-07-04 扶桑薬品工業株式会社 ｃＡＭＰおよびアデニル酸シクラ―ゼの酵素的蛍光定量アッセイ
NZ514305A (en) 1999-04-16 2004-01-30 Univ Yale eNOS mutations useful for gene therapy and therapeutic screening
ATE374820T1 (de) 1999-10-26 2007-10-15 Secr Defence Mutantenluciferase
JP2003513651A (ja) 1999-11-08 2003-04-15 ルミテック・（ユーケー）・リミテッド 細胞培養物中の微生物のアッセイ
WO2001053513A1 (fr) 2000-01-17 2001-07-26 Satake Corporation Systemes et procede de reaction de regeneration atp permettant d'examiner le nucleotide d'adenine, procede de detection d'arn et procede d'amplification d'atp
US6265179B1 (en) 2000-02-01 2001-07-24 Molecular Probes, Inc. Detection of phosphate using coupled enzymatic reactions
GB0030727D0 (en) 2000-12-15 2001-01-31 Lumitech Uk Ltd Methods and kits for detecting kinase activity
US20080050762A1 (en) 2001-02-13 2008-02-28 Corey Michael J Methods and compositions for coupled luminescent assays
US6811990B1 (en) 2001-02-13 2004-11-02 Michael J. Corey Methods and compositions for coupled luminescent assays
JP3992467B2 (ja) 2001-09-05 2007-10-17 独立行政法人科学技術振興機構 ｃＡＭＰ及びｃＧＭＰを高感度で測定し得るサンプルの同時調製法
US7268229B2 (en) 2001-11-02 2007-09-11 Promega Corporation Compounds to co-localize luminophores with luminescent proteins
EP1546374B1 (en) 2002-09-06 2010-11-10 Promega Corporation Method for detecting transferase enzymatic activity
GB2394286A (en) 2002-09-17 2004-04-21 Innovab Ltd An assay for adp using an adp binding protein
AU2003267245C1 (en) 2002-09-20 2008-05-29 Promega Corporation Luminescence-based methods and probes for measuring cytochrome P450 activity
WO2004068115A2 (en) 2003-01-30 2004-08-12 Bellbrook Labs, Llc Assay method for group transfer reactions
AU2003212402A1 (en) 2003-03-20 2004-10-11 Csi Biotech Oy Amp ligation assay (ala)
US8476034B2 (en) 2003-07-10 2013-07-02 General Atomics Methods and compositions for assaying homocysteine
US7338775B1 (en) 2005-02-09 2008-03-04 Myriad Genetics, Inc. Enzyme assay and use thereof
US7410755B2 (en) 2005-02-22 2008-08-12 Discoverx ADP detection using an enzyme-coupled reaction
JP2008531025A (ja) 2005-02-28 2008-08-14 ニコメッド ゲゼルシャフト ミット ベシュレンクテル ハフツング Ｐｄｅ１１モジュレーターの同定方法
JP5350785B2 (ja) 2005-05-31 2013-11-27 プロメガ コーポレイション 分子または状態を検出するための発光性化合物および蛍光性化合物、ならびに方法
CN101784274A (zh) 2007-06-15 2010-07-21 米申制药公司 抑制水肿因子和腺苷酸环化酶的方法和组合物
US20090075309A1 (en) 2007-09-06 2009-03-19 Gambhir Sanjiv S Bisdeoxycoelenterazine derivatives, methods of use, and BRET2 systems
US8420341B2 (en) * 2007-12-14 2013-04-16 Genewiz Inc., Suzhou Methods for measuring ADP
CN102159948B (zh) * 2008-07-22 2014-06-25 普罗美加公司 基于adp检测的发光磷酸转移酶或atp水解酶测定
EP2771480B1 (en) 2011-10-28 2016-12-14 Promega Corporation Methods for detecting adenosine monophosphate in biological samples

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
None *

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JP6510041B2 (ja) 2019-05-08
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US9677117B2 (en) 2017-06-13
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